Umbilical Cord Blood Natural Killer Research Articles

Exploring NK cell receptor dynamics in paediatric leukaemias: implications for immunotherapy and prognosis

AbstractObjectivesImmunotherapies targeting natural killer (NK) cell receptors have shown promise against leukaemia. Unfortunately, cancer immunosuppressive mechanisms that alter NK cell phenotype prevent such approaches from being successful. The study utilises advanced cytometry to examine how cancer immunosuppressive pathways affect NK cell phenotypic changes in clinical samples.MethodsIn this study, we conducted a high‐dimensional examination of the cell surface expression of 16 NK cell receptors in paediatric patients with acute myeloid leukaemia and acute lymphoblastic leukaemia, as well as in samples of non‐age matched adult peripheral blood (APB) and umbilical cord blood (UCB). An unsupervised analysis was carried out in order to identify NK cell populations present in paediatric leukaemias.ResultsWe observed that leukaemia NK cells clustered together with UCB NK cells and expressed relatively higher levels of the NKG2A receptor compared to APB NK cells. In addition, CD56dimCD16+CD57− NK cells lacking NKG2A expression were mainly absent in paediatric leukaemia patients. However, CD56br NK cell populations expressing high levels of NKG2A were highly represented in paediatric leukaemia patients. NKG2A expression on leukaemia NK cells was found to be positively correlated with the expression of its ligand, suggesting that the NKG2A‐HLA‐E interaction may play a role in modifying NK cell responses to leukaemia cells.ConclusionWe provide an in‐depth analysis of NK cell populations in paediatric leukaemia patients. These results support the development of immunotherapies targeting immunosuppressive receptors, such as NKG2A, to enhance innate immunity against paediatric leukaemia.

High dimensional analysis reveals distinct NK cell subsets but conserved response to stimulation in umbilical cord blood and adult peripheral blood.

Growing interest surrounds adoptive cellular therapies utilizing Natural Killer (NK) cells, which can be obtained from various sources, including umbilical cord blood (UCB) and adult peripheral blood (APB). Understanding NK cell receptor expression and diversity in such cellular sources will guide future therapeutic designs. We used a 20-color flow cytometry panel to compare unstimulated and cytokine-activated UCB and APB NK cells. Our analysis showed that UCB NK cells express slightly higher levels of the immune checkpoints PD-1, TIGIT, and CD96 compared to their APB counterparts. Unsupervised hierarchical clustering and dimensionality reduction analyses revealed enrichment in CD56neg as well as mature NKp46neg and CD56+ CD16+ NK cell populations in UCB whereas CD57+ terminally differentiated NK cells with variable expression of KIRs and CD16 were found in APB. These populations were conserved following stimulation with IL-12, IL-15, and IL-18. Cytokine stimulation was associated with the downregulation of TIGIT and CD16 on multiple NK cell subsets in UCB and APB. Among UCB CD16- NK cell populations, TIGIT+ NK cells produced more IFN-γ than their TIGIT- counterparts. Our data demonstrate higher immune checkpoint expression on UCB NK cells compared to APB. However, the expression of TIGIT immune checkpoint is not indicative of NK cell exhaustion.

Comparative Study of the Two High-Efficient Strategies for in vitro Generation of Human Umbilical Cord Blood-derived Natural Killer Cells

To explore the similarities and variations of biological phenotype and cytotoxicity of human umbilical cord blood natural killer cells (hUC- NK) after human umbilical cord blood-derived mononuclear cells (hUC-MNC) activated and expanded by two in vitro high-efficient strategies. Umbilical cord blood mononuclear cells (MNC) from healthy donor were enriched by Ficoll-based density gradient centrifugation. Then, the phenotype, subpopulations, cell viability and cytotoxicity of NK cells derived from Miltenyi medium (denoted as M-NK) and X-VIVO 15 (denoted as X-NK) were compared using a "3IL" strategy. After a 14-day's culture, the contents of CD3-CD56+ NK cells were elevated from 4.25%±0.04% (d 0) to 71%±0.18% (M-NK) and 75.2%±1.1% (X-NK) respectively. Compared with X-NK group, the proportion of CD3+CD4+ T cells and CD3+CD56+ NKT cells in M-NK group decreased significantly. The percentages of CD16+, NKG2D+, NKp44+, CD25+ NK cells in X-NK group was higher than those in the M-NK group, while the total number of expanded NK cells in X-NK group was half of that in M-NK group. There were no significant differences between X-NK and M-NK groups in cell proliferation and cell cycle, except for the lower percentage of Annexin V+ apoptotic cells in M-NK group. Compared with X-NK group, the proportion of CD107a+ NK cells in M-NK group were higher under the same effector-target ratio (E∶T) (P<0.05). The two strategies were adequate for high-efficient generation of NK cells with high level of activation in vitro, however, there are differences in biological phenotypes and tumor cytotoxicity.

An Injectable Epigenetic Autophagic Modulatory Hydrogel for Boosting Umbilical Cord Blood NK Cell Therapy Prevents Postsurgical Relapse of Triple-Negative Breast Cancer.

Triple‐negative breast cancer (TNBC) exhibits resistance to conventional treatments due to the presence of cancer stem cells (CSCs), causing postsurgical relapse and a dismal prognosis. Umbilical cord blood natural killer (UCB‐NK) cell‐based immunotherapy represents a promising strategy for cancer treatment. However, its therapeutic efficacy is greatly restrained by downregulation of the NK cell activation ligand MHC class I‐related chain A/B (MICA/B) and autophagy‐mediated degradation of NK cell‐derived granzyme B (GZMB) in CSCs. Herein, it is demonstrated that suberoylanilide hydroxamic acid (SAHA) epigenetically downregulates let‐7e‐5p and miR‐615‐3p to increase MICA/B expression and that 3‐methyl adenine (3MA) inhibits autophagy‐mediated GZMB degradation, thereby sensitizing breast CSCs to UCB‐NK cells. Then, an injectable hydrogel is designed to codeliver SAHA and 3MA to enhance UCB‐NK cell infusion efficacy in TNBC. The hydrogel precursors can be smoothly injected into the tumor resection bed and form a stable gel in situ, allowing for a pH‐sensitive sustained release of SAHA and 3MA. Moreover, UCB‐NK cell infusion in combination with the hydrogel efficiently controls postsurgical relapse of TNBC. In addition, the hydrogel exhibits good hemostasis and wound‐healing functions. Therefore, the work provides proof of concept that an injectable epigenetic autophagic modulatory hydrogel augments UCB‐NK cell therapy to combat postsurgical relapse of TNBC.

Abstract LB211: Tumor-reactive and anti-PD-L1 co-stimulated killer cells (TRACK-NK) for immunotherapy of non-small cell lung cancer

Abstract Non-small cell lung cancer (NSCLC) remains one of the major causes of cancer related mortality worldwide, yet clinical advances with checkpoint inhibitors suggest that an anti-tumor immune response can be harnessed to prolong survival in NSCLC patients. We recently described the identification of a tumor reactive natural killer (NK) cell population by its in vivo upregulation of PD-L1 in the presence of tumor, referred to here as TRACK-NK cells. PD-L1(+) NK cells are more potent than PD-L1(-) NK cells against tumor cell targets. Further, the administration of anti-PD-L1 antibody (atezolizumab) functions as an immune agonist on TRACK-NK cells resulting in significantly enhanced degranulation (P &amp;lt; 0.001) and secretion of IFN-gamma (P &amp;lt; 0.001) in the presence of tumor. Here we investigated the functional activity and product safety of allogeneic, off-the-shelf TRACK-NK cells retrovirally transduced to express soluble (s) IL-15 to treat NSCLC without or with the addition of atezolizumab. TRACK-NK cells derived from umbilical cord blood NK cells were expanded over 1,000-fold ex-vivo in 17 days with ~50% of the final product engineered to express sIL15 and cytokine-activated to express PD-L1. The phenotype of the final TRACK-NK cell product showed a significant increase in the expression of PD-L1, CD25 and CD69 when compared to non-transduced (P &amp;lt; 0.01) or NK cells transduced to express sIL15 without cytokine activation (s15 NK cells) (P &amp;lt; 0.01). Following cryopreservation, our thawed TRACK-NK cell product demonstrated high recovery (&amp;gt;85%) and viability (&amp;gt;80%). In a Real Time Cell Analysis (RTCA) in vitro cytotoxicity assay against a human NSCLC cell line (A549), TRACK-NK cells were 10 times more potent than non-transduced (NT) NK cells (P &amp;lt; 0.001) and 2 times more potent than s15 NK cells (P &amp;lt; 0.01). To assess the efficacy of our frozen, allogeneic off-the-shelf TRACK-NK cells in vivo, we utilized immunodeficient mice bearing human A549 metastatic NSCLC. Intravenous infusion of TRACK-NK cells resulted in significantly more suppression of luciferase-A549 NSCLC growth in mice compared to mice treated with NT NK cells (P &amp;lt; 0.05) or s15 NK cells (P &amp;lt; 0.01). In terms of dose response, 1 × 107 TRACK-NK cells per dose delivered every other day for a total of 4 doses showed significantly better tumor control compared to a dose of 5 × 106 TRACK-NK cells (P &amp;lt; 0.001) but similar control compared to 2 × 107 TRACK-NK cells. The combination of TRACK-NK cells plus atezolizumab showed better control of NSCLC growth in vivo compared to TRACK-NK cells alone (P &amp;lt; 0.01). Finally, we evaluated the safety of our TRACK-NK product in immunodeficient mice without tumor engraftment and did not observe any significant change in body weight, body temperature, liver enzymes, kidney function, or blood counts compared to mice treated with vehicle control. No cytokine release syndrome was observed. In summary, our studies using human allogeneic off-the-shelf TRACK-NK cells proved to be safe and non-toxic, demonstrated enhanced cytotoxicity against NSCLC in vitro and enhanced control of tumor growth in vivo, further improved with atezolizumab. As the intravenous infusion of activated NK cells naturally traffic to lung, our TRACK-NK platform will move forward to the clinic for the treatment of relapsed and refractory NSCLC patients. Citation Format: Ting Lu, Anthony G. Mansour, Rui Ma, Christian Bustillos, Zhiyao Li, Shoubao Ma, Zhenlong Li, Hanyu Chen, Kun-Yu Teng, Jianying Zhang, Michael A. Caligiuri, Jianhua Yu. Tumor-reactive and anti-PD-L1 co-stimulated killer cells (TRACK-NK) for immunotherapy of non-small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr LB211.

Optimizing NK Cell-Based Immunotherapy in Myeloid Leukemia: Abrogating an Immunosuppressive Microenvironment.

Natural killer (NK) cells are prominent cytotoxic and cytokine-producing components of the innate immune system representing crucial effector cells in cancer immunotherapy. Presently, various NK cell-based immunotherapies have contributed to the substantial improvement in the reconstitution of NK cells against advanced-staged and high-risk AML. Various NK cell sources, including haploidentical NK cells, adaptive NK cells, umbilical cord blood NK cells, stem cell-derived NK cells, chimeric antigen receptor NK cells, cytokine-induced memory-like NK cells, and NK cell lines have been identified. Devising innovative approaches to improve the generation of therapeutic NK cells from the aforementioned sources is likely to enhance NK cell expansion and activation, stimulate ex vivo and in vivo persistence of NK cells and improve conventional treatment response of myeloid leukemia. The tumor-promoting properties of the tumor microenvironment and downmodulation of NK cellular metabolic activity in solid tumors and hematological malignancies constitute a significant impediment in enhancing the anti-tumor effects of NK cells. In this review, we discuss the current NK cell sources, highlight ongoing interventions in enhancing NK cell function, and outline novel strategies to circumvent immunosuppressive factors in the tumor microenvironment to improve the efficacy of NK cell-based immunotherapy and expand their future success in treating myeloid leukemia.

Umbilical cord blood NK cells offer multiple advantages for cancer immunotherapy: lessons learned from Glycostem’s orphan drug oNKord®

Adoptive transfer of natural killer (NK) cells has been demonstrated as an efficacious and safe treatment option in patients with cancer. For clinical use, these potent immune cells can be directly isolated or generated from several sources using a variety of protocols. Increasing evidence indicates that NK cell products prepared from hematopoietic stem and progenitor cells isolated from umbilical cord blood have many advantages. These include a highly cytotoxic activity against hematologic and solid malignancies, a minimal risk of adverse events, including graft-versus-host disease, and wide availability and applicability. The objective of this review is to provide an oversight of the various NK cell products and to discuss their advantages and disadvantages for cancer immunotherapy.

Exploring the NK cell platform for cancer immunotherapy.

Natural killer (NK) cells are cytotoxic lymphocytes of the innate immune system that are capable of killing virally infected and/or cancerous cells. Nearly 20 years ago, NK cell-mediated immunotherapy emerged as a safe and effective treatment approach for patients with advanced-stage leukaemia. Subsequently, the field of NK cell-based cancer therapy has grown exponentially and currently constitutes a major area of immunotherapy innovation. In general, the development of NK cell-directed therapies has two main focal points: optimizing the source of therapeutic NK cells for adoptive transfer and enhancing NK cell cytotoxicity and persistence in vivo. A wide variety of sources of therapeutic NK cells are currently being tested clinically, including haploidentical NK cells, umbilical cord blood NK cells, stem cell-derived NK cells, NK cell lines, adaptive NK cells, cytokine-induced memory-like NK cells and chimeric antigen receptor NK cells. A plethora of methods to augment the cytotoxicity and longevity of NK cells are also under clinical investigation, including cytokine-based agents, NK cell-engager molecules and immune-checkpoint inhibitors. In this Review, we highlight the variety of ways in which diverse NK cell products and their auxiliary therapeutics are being leveraged to target human cancers. We also identify future avenues for NK cell therapy research.

Patients Lacking a KIR-Ligand of HLA Group C1 or C2 Have a Better Outcome after Umbilical Cord Blood Transplantation.

Donor natural killer (NK) cells can destroy residual leukemic cells after allogeneic hematopoietic stem cell transplantation. This effect is based on the interaction of killer-cell immunoglobulin-like receptors (KIR) of donor NK cells with ligands of the major histocompatibility complex found on the surface of the target cells. HLA-C1 subtypes provide the ligand for KIR2DL2 and KIR2DL3 and the HLA-C2 subtypes for KIR2DL1. We have studied the probability of relapse (PR) after single-unit unrelated cord blood transplantation (UCBT) in relation to the potential graft-vs.-leukemia effect mediated by NK cells present in the umbilical cord blood (UCB) by analyzing KIR-ligand and HLA-C typing of the receptor. Data from 33 consecutive patients given a single unit UCBT were included. We have considered two groups of patients based on the absence or the presence of one of the C-ligands for inhibitory KIR and the incompatibility HLA-C1/2 between UCB and patients. Group 1 (n = 21): the patient lacks a C-ligand for inhibitory KIR present in UCB NK cells, i.e., patients homozygous C1/C1 or C2/C2. Group 2 (n = 12): patients heterozygous C1/C2 in which KIR-mediated graft-vs.-leukemia effect is not expected (presence of both C ligands for inhibitory KIR in the receptor). With a median follow-up post-UCBT of 93 months, patients with absence of a C-ligand for inhibitory KIRs (Group 1) showed a lower actuarial PR than patients with both C-ligands (group 2): 21 ± 10 vs. 68 ± 18% at 2 year and 36 ± 13 vs. 84 ± 14% at 5 years (p = 0.025), respectively. In patients with acute lymphoblastic leukemia, the 2-year PR was 36 ± 21% for group 1 and 66 ± 26% for 2 (p = 0.038). Furthermore, group 1 had a lower incidence of grades II–IV acute graft-vs.-host disease (p = 0.04). In the setting of UCBT, the absence of a C-ligand (C1 or C2) of inhibitory KIR in the patient is associated with lower PR, which is probably due to the graft-vs.-host leukemia effect caused by UCB NK cells that lack a ligand for the inhibitory KIR 2DL1/2DL2/2DL3.

Umbilical Cord Blood Natural Killer Cells, Their Characteristics, and Potential Clinical Applications

Natural killer (NK) cells are lymphocytes of the innate immune system able to kill different targets such as cancer cells and virally infected cells without prior activation making then attractive candidates for cancer immunotherapy. Umbilical cord blood (UCB) has become a source of hematopoietic stem cells for transplantation but as we gain a better understanding of the characteristics of each immune cell that UCB contains, we will also be able to develop new cell therapies for cancer. In this review, we present what is currently known of the phenotype and functions of UCB NK cells and how these cells could be used in the future for cancer immunotherapy.

Effect of Influenza A Infection on Umbilical Cord Blood Natural Killer Function Regulation With Interleukin-15

Influenza A is a major pathogen of humans and has the potential to cause worldwide pandemics. Natural killer (NK) cells are important effector cells in the innate immune response against viruses, including influenza A. Infants are more susceptible to severe influenza A viral infection, possibly attributed in part to their defective NK function. We compared the NK responses to influenza using umbilical cord blood (UCB) and adult peripheral blood (APB) mononuclear cells and purified NK cells. Influenza A induced dose-dependent apoptosis of NK cells with down-regulation of NKp46 expression, which was more pronounced in UCB. Both UCB and APB NK cells responded to influenza infection by up-regulating CD69 and CD107a expression, a process further enhanced by interleukin (IL) 15. Influenza exposure also down-regulated perforin expression and K562 cytotoxicity in UCB NK cells, which was partially restored by IL-15. The production of interferon (IFN) γ and tumor necrosis factor (TNF) α by NK cells in responding to influenza was further enhanced by IL-15. Our findings show differential NK responses between newborns and adults. IL-15 may be beneficial in combating influenza by enhancing cytotoxic function and IFN-γ production.

Cytotoxic Function of Umbilical Cord Blood Natural Killer Cells: Relevance to Adoptive Immunotherapy

Decreased graft-versus-host disease (GVHD), ease of accessibility, and sustained engraftment encourage the use of umbilical cord blood (UCB) as an alternative source to bone marrow for immune reconstitution in children with leukemia. Natural killer (NK) cells rapidly expand after stem cell transplantation and are important for regulating GVHD and providing graft-versus-leukemia (GVL) effects. This review highlights the phenotypic and functional differences between UCB NK cells and adult peripheral blood (APB) NK cells, and discusses the possible therapeutic benefit of using UCB NK cells for adoptive immunotherapy in leukemia. Alloreactive NK cells show potent cytotoxic activities against human leukocyte antigen (HLA)-nonidentical leukemic cells and reduce leukemia relapses. The higher numbers of NK progenitors in UCB makes it a convenient source for ex vivo expansion of UCB NK cells for posttransplant treatment. UCB NK cells readily respond to interleukin-15, which may greatly enhance their antitumor effect. Activation and expansion protocols for UCB NK cells are currently being developed.

Interleukin (IL)-15 in combination with IL-2, fms-like tyrosine kinase-3 ligand and anti-CD3 significantly enhances umbilical cord blood natural killer (NK) cell and NK-cell subset expansion and NK function

Interleukin (IL)-15 and fms-like tyrosine kinase-3 (FLT-3) are crucial factors for the development of human and murine natural killer (NK) cells. Previously, we have demonstrated significant ex vivo expansion and activation of unrelated cord blood (UCB) NK cells with an antibody/cytokine cocktail consisting of anti-CD3 + IL-2 + IL-12 + IL-7 and anti-CD3 + IL-2 + IL-12 + IL-18. In the current experiments, we investigated the effects of short-term culture with anti-CD3 + IL-2 + FLT-3 + IL-15 on cord blood (CB) NK cell and NK-cell subset expansion and function. CB mononuclear cells were cultured for 48 h in AIM-V media or AIM-V + IL-2 (5 ng/mL) + anti-CD3 (50 ng/mL) + FLT-3 (50 ng/mL) ± escalating doses of IL-15 (1, 10 or 100 ng/mL). Flow cytometric analysis was performed using various fluorescent-conjugated monoclonal antibodies. In vitro cytotoxicity was determined with a standard europium assay against K562 and Daudi cells. There was a 4.8-fold significant increase in NK-cell population (CD3(-)/16(+)/56(+); P < 0.03), 21-fold significant increase in CD3(-)/56(+)/158a(+) (KIR2DL1/S1; P < 0.002), 46-fold significant increase in CD3(-)/56(+)/158b(+) (KIR2DL1/S2; P < 0.002) and 11.5-fold significant increase in CD3(-)/56(+)/NKB1(+) (KIR3DL1; P < 0.01). We also noted a significant increase in both NK and lymphokine-activated killer (LAK) cytotoxicity with IL-2 + anti-CD3 + FLT-3 + IL-15 (100 ng/mL) compared with IL-2 + anti-CD3 + FLT-3 and media alone against K562 (P < 0.01) and Daudi (P < 0.001), respectively. We have demonstrated a significant increase in UCB NK cells and NK cells expressing a variety of killer immunoglobulin-like receptor (KIR) receptors after short-term culture with anti-CD3, IL-2, FLT-3 and IL-15. Furthermore, there was a significant increase in in vitro NK/LAK cell cytotoxicity.

The phenotypic and functional characteristics of umbilical cord blood and peripheral blood natural killer cells

Allogeneic hematopoietic cell transplantation can be curative for patients with high-risk acute leukaemia. Umbilical cord blood (UCB) is an increasingly used source of allogeneic stem cells for patients who are in need of a transplant, but do not have a sibling donor. This review highlights the similarities and differences between the natural killer (NK) cells obtained from adult peripheral blood (PB) and UCB. These two cell sources show similar percentages of NK cells, including the major CD56(dim) and CD56(bright) subpopulations. UCB also contains an additional CD56-CD16+ subset, not typically found in PB. In addition, there are a number of progenitor cell populations in UCB that can give rise to NK cells. Some studies showed that UCB NK cells express a relatively higher percentage of inhibitory receptors (CD94/NKG2A and killer-cell immunoglobulin-like receptors) and less adhesion molecules. Resting UCB NK cells also show significantly less cytotoxicity compared to PB NK cells. However, following cytokine stimulation, the cytotoxicity of UCB NK cells can be rapidly increased to levels that are comparable to PB NK cells. Activation and expansion protocols for UCB NK cells are briefly reviewed. Lastly, we outline the early use of UCB NK cells in clinical trials.

Alterations in the Umbilical Cord Blood NK Cells Related to the Mode and the Time of Delivery.

Natural killer (NK) cells take part in the early immunological response to infection. Their lower cytotoxic activity in the neonates, especially premature ones, compared to children and adults, is assumed to be one of the factors responsible for high susceptibility to infections. Moreover, alterations in every components of immune response during anesthesia and surgery have been suggested. The numbers of natural killer cells are decreased postoperatively. The aim of the study was to estimate the influence of the mode and time of delivery on the number of leukocytes, number and percentage of lymphocytes and natural killer (NK) cells. The NK cells were examined by the three-color flow cytometry with the use of monoclonal antibodies of Becton Dickinson in the following study groups: (1) full-term neonates born by normal spontaneous vaginal delivery (n=19); (2) preterm neonates born by normal spontaneous vaginal delivery (n=15); (3) full-term neonates born by elective cesarean section under epidural anesthesia (n=23); (4) preterm neonates born by cesarean section under epidural anesthesia (n=22). The number of leukocytes was similar in all examined neonates. The numbers of leukocytes were lower albeit not significantly in preterm neonates born by cesarean section. The numbers of lymphocytes were also similar in all examined neonates but the percentage of lymphocytes was higher in the preterm neonates than in the full-term ones (p<0,05). The number and percentage of natural killer (NK) cells were higher in the neonates born by normal spontaneous vaginal delivery both full-term and preterm ones. The significant lowest value of NK cells was in the preterm neonates born by cesarean section under epidural anesthesia. These results suggest that either mode of delivery or time of delivery might influence the NK cell numbers in the umbilical cord blood of the neonates.

Comparison of Surface Phenotype, Conjugation, and Regulatory Proteins within Natural Killer (NK) Populations Derived from Umbilical Cord Blood (UCB) and Adult Blood (AB).

NK cells mediate innate immune system responses in early defense through cytolysis and the production of regulatory cytokines and chemokines. We sought to investigate whether there are differentiating characteristics of NK cells derived from neonatal peripheral blood and adult peripheral blood. These analyses included surface phenotype, confocal imaging of lipid rafting, measurement of target conjugation, and Western blot analyses of regulatory proteins. Immunophenotyping of UCB grafts (n=40) revealed 14.1% (SEM 0.9%) of UCB mononuclear cells (MNC) expressed NK phenotypes (CD56+ CD3−). Lipid raft migration within NK cells was achieved through stimulation with K562 targets via brief centrifugation. One million NK and target cells were placed in a 1:1 and 1:2 respective ratio. Lipid rafts were stained with anti-cholera toxin B and cholera-toxin B Alexa Flour-594, which bind to cellular surfaces via the pentasaccharide chain of ganglioside GM1. In addition, K562 plasma membranes were stained with concanavalin A (FITC), which selectively binds to alpha-mannopyranosyl and alpha-glucopyranosyl residues. In order to excite appropriate wavelengths and acquire pictures, Argon (488 nm) and Helium-Neon (543 nm) lasers were utilized in conjunction with a Zeiss 510 laser scanning confocal microscope. Lipid raft recruitment and formation of immunological synapse of stimulated AB and UCB NK cells were observed in the presence of K562 targets. These confocal images were verified by quantification of NK/target conjugates through flow cytometry to assess the percent fold change of conjugation between AB NK cells co-cultured with self targets in comparison to third party HLA (KIR) disparate targets. The percentage of conjugation between AB NK cells in third party HLA disparate targets compared with HLA matched MNC targets demonstrated an average increase in fold change of 7.2 (1.6) (n=7). Next, NFATc2 (NFAT1) protein expression in UCB and AB was measured by Western blot (anti-NFAT1, BD Transduction Laboratories, San Diego, CA). This transcription factor is of particular interest due its known regulation of cytokines and chemokines including IFN-gamma and TNF-alpha. NK cells were isolated from UCB or AB by magnetic separation (AutoMacs, Miltenyi Biotech, Auburn, CA). Surface phenotype and purity were verified by flow cytometry including CD3, CD4, CD8, CD19, and CD56. Average purity was 81.5% (3.7%) CD56+CD3− NK (n=4) from UCB MNC and 76.9% (2.9%) (n=4) from AB MNC. Western blotting demonstrated the intensity of NFATc2 (NFAT1) protein expression in AB NK relative to UCB NK was 1495, showing higher expression in AB NK.Ongoing experiments include further analysis of UCB NK cells by confocal imaging and measurement of rates of conjugation by multi-color flow cytometry as well as measurement of regulatory proteins including Zap-70, LcK, Syk, PKC-theta, and LAT comparing UCB and AB NK. In conclusion, neonatal peripheral blood CD56+CD3− NK differ from their adult counterpart in expression of transcription regulatory proteins including NFATc2 (NFAT1), as well as rates of conjugation and lipid rafting. These observed differences in innate immune responses of neonatal NK cells compared with that of adults has important implications in immune tolerance.

Human umbilical cord blood NK T cells kill tumors by multiple cytotoxic mechanisms

Natural killer (NK) T cells are restricted by CD1d and play an important role in the rejection of malignant tumors, but how kill these tumors is unclear. To investigate this, we cultured Vα24 +CD4 + NK T cells in human umbilical cord blood, which was enriched by immunomagnetic beads. In short-term (4 h) cytotoxicity assays, the NK T cells could kill only those targets expressing CD1d. In longer cytotoxicity assays (20 h), however, the NK T cells were able to kill all the tumors, regardless of CD1d expression. When each of the perforin, Fas-FasL, and TNF-α cytotoxic mechanisms were blocked, it was apparent that perforin killing dominated in both the short- and long-term assays. In the short-term assay, perforin killing required that the targets expressed CD1d, but killing was more efficient if Fas was present because then the Fas-FasL mechanism was also used. Thus, cells that lacked Fas and CD1d and were not killed in the 4-h assay, were instead lysed in 20-h assay through a combination of perforin and TNF-α killing. NK T cells can kill tumor targets by perforin, Fas-FasL, and TNF-α mechanisms. TNF-α killing requires longer contact between effectors and targets, suggesting that TNF-α acts by enhancing perforin killing.