Cord Blood Natural Killer Cells Research Articles

Genetic Engineering and Significant Ex-Vivo Expansion of Cord Blood Natural Killer Cells: Implications for Post-Transplant Adoptive Cellular Immunotherapy

Natural killer (NK) cells are a critical component of both the innate and adaptive human immune response (Caligiuri et al, Blood 2008). Tumor target cell recognition by NK cells is a highly regulated and complex set of processes which are controlled by the balance between inhibitory and activating signals through the binding of a variety of ligands on tumor target cells by several distinct subtypes of NK cell receptors (Bryceson YT et al, Immun Rev, 2006). The major limitations of the use of NK cells in adoptive tumor cellular immunotherapy include lack of tumor recognition and activation and/or limited numbers of viable and functionally active NK cells (Shereck/Cairo et al, Pediatr Blood Cancer, 2007). To circumvent these limitations, methods to expand and/or activate peripheral blood NK cells have been developed. Over the past decade cord blood (CB) has been increasingly utilized as an alternative to peripheral blood for allogeneic stem cell transplantation (Cairo et al, Blood, 1997). We recently reported the successful expansion and functional activation of CB NK cells by ex-vivo cellular engineering with a cocktail of antibody and cytokines (Ayello/Cairo et al, BBMT, 2006). In addition, our group has had a major interest in the diagnosis, treatment and biology of childhood CD20+ B-NHL; and have identified subgroups of patients with a significantly poorer prognosis despite aggressive multiagent chemotherapy (Cairo et al, Blood, 2007). In this study we sought to to develop an adoptive cellular immunotherapy strategy to overcome chemotherapy drug resistant childhood B-NHL. Freshly isolated CB mononuclear cells (CBMC) were cultured with modified K562 cells expressing membrane bound IL15 and 4-1BB ligand (K562-mbIL15-41BBL; Imai et al, Blood, 2005). After irradiation with 100Gy, K562- mbIL15-41BBL cells were incubated in a 1:1 ratio with CBMC + 10 IU/mL rhIL-2 for 7–14 days. CD3 and CD56 expression was determined by flow cytometry at Days 0, 7 and 14. On Day 0, CBMC included a population of NK cells expressing CD56 of 3.9% ± 1.3% and CD3+ T cells of 48.3% ± 3.9%. After 7 days of culture with K562-mbIL15- 41BBL cells the percentage of CD56+/CD3− NK cells increased to 71.7% ± 3.9%, as compared to 9.7% ± 2.4% in cultures with media alone and 42.6% ± 5.9% in cultures with wild-type K562 cells (p<0.01). There was also a significant decrease in the percentage of T cells in cultures with the modified K562 cells compared to wild-type K562 and media alone (15.2% ± 2.2% vs 35.4% ± 4.4% vs 51.2% ± 7.1%, p<0.001). Overall, the percent of NK cells after 7 days of culture with K562-mbIL15-41BBL was 3374% ± 385% of the input cell number, i.e. an approximate 35-fold increase. This is significantly increased compared to culture with wild-type K562 (1771% ± 300%, p<0.05). On Day 14, there remained a significant difference in NK cell populations between CBMC incubated with modified K562 cells compared to wild-type K562 cells (62.0% ± 2.1% vs 27.9% ± 2.4%, p<0.001), and compared to media alone (5.5% ± 0.4%, p<0.001) but no further increase from Day 7. Expansion of NK cells using genetically modified K562 cells as a stimulus produced significantly higher numbers of NK cells than those previously observed using a cocktail of antibody and cytokines as a stimulus (Ayello/Cairo et al, BBMT, 2006): (71.7% ± 3.9% NK cells on Day 7 with modified K562 vs 33.9% ± 8.7% with AB/CY, p=0.0004). In summary, we have demonstrated CBMC can be stimulated by K562 cells expressing membrane bound IL15 and 4-1BB ligand (K562-mbIL15-41BBL) resulting in specific expansion of CB NK cells similar or higher than the expansion that can be obtained with peripheral blood. The method described here provides a means to promote CB NK-mediated cellular cytotoxicity for use in the post-transplant setting while minimizing the risk of graft-versus-host disease.

Umbilical Cord Mesenchymal Stem Cells Increase Expansion of Cord Blood Natural Killer Cells

Natural killer (NK) cell-mediated cytotoxicity can control leukemia relapse while protecting patients from graft-versus-host disease (GVHD) after allogeneic stem cell transplant. Cord blood (CB) is rich in NK cell progenitors with similar properties of proliferation and cytotoxicity as adult blood NK cells. Hence, it is attractive to expand and potentially utilize these cells for adoptive immunotherapy. In this study, CB mononuclear cells were CD3-depleted by immunomagnetic microbead selection to remove T cells. This CD3dep CB-MNC fraction was then plated for ex vivo expansion, with or without a feeder layer of irradiated umbilical cord mesenchymal stem cells (UC-MSC), with or without cytokines that have been shown to be critical for NK expansion: IL-2, IL-15, IL-3, and FLT-3L. At an average of 2 weeks of culture, there was significantly higher expansion (64.7 ± 8.4-fold) of CD56+/CD3− NK cells in the presence of the UC-MSC feeder layer and cytokines compared to controls (no increase with feeder layer only and 6.4 ± 1.5-fold increase with cytokines only, P < .05). Contact between CD3dep CB-MNC cells and UC-MSC augmented NK expansion. The combination of all 4 cytokines was superior to IL-2 alone or 2 cytokines combinations: mean 64.7 ± 8.4-fold expansion with 4 cytokines combination versus IL-2 alone, IL-2 + FLT-3L, IL-2 + IL-15 or IL-2 + IL-3 (12.2 ± 2.0, 14.4 ± 2.4, 10.4 ± 4.1, 25.2 ± 8.1 respectively). We also observed that only fresh CD3dep CB-MNC preparations could be expanded reliably, whereas frozen and thawed CD3dep CB-MNC cells did not expand consistently (mean fold increase 6.5 ± 3.2). Cytotoxicity of expanded NK cells was compared with NK cells from fresh and overnight IL-2 activated CD3dep CB-MNC. Whereas fresh cells displayed no discernible killing, strong cytotoxicity against K562, Raji, REH, and SUP-B15 cells lines was noted after overnight activation in IL-2. Cytotoxicity of expanded NK cells against Raji, REH, and SUP-B15 was lower, which, however, correlated with a predominant expansion of CD56+/CD16− cells known to have less cytolytic activity than CD56+/CD16+. To test the transfection efficiency in NK cells, fresh or expanded CD3dep CB-MNC cells were electroporated with either DNA or mRNA constructs for GFP. DNA had a low transfection efficiency (<10%), whereas the one for mRNA reached 52%, but at the cost of significant cell death. Our results suggest that CB NK cell progenitors can be expanded to obtain large numbers by using an irradiated feeder of UC-MSC. They maintain an elevated cytotoxic profile, and may be genetically manipulated—all characteristics that make them suitable for cellular therapies.

Effect of cyclosporin A on interleukin-15-activated umbilical cord blood natural killer cell function

Interleukin (IL)-15-activated natural killer (NK) cells may provide a graft-versus-leukemia (GvL) effect post-umbilical cord blood (CB) transplantation. The effect of cyclosporin A (CsA), a calcineurin-inhibitor used for prophylaxis of graft-versus-host disease (GvHD), on IL-15-mediated activation, cytotoxic function and target-induced apoptosis of CB NK cells, was examined in comparison with adult peripheral blood (APB) NK cells. CsA was added to anti-CD3+/-IL-15-stimulated CB and APB mononuclear cells (MNC) for a 5-day incubation. CD3- CD56+ NK cell recovery was determined by flow cytometric analysis. Magnetic bead-purified CB and APB NK cells were stimulated with IL-15 for 18 h under the influence of CsA. NK activation (CD69), K562 cytotoxicity and NK-K562 interactions (CD54, perforin and annexin-V expression 4 h following contact with K562 cells) were assessed by flow cytometry. CsA decreased CD3- CD56+ NK cell recovery in anti-CD3-stimulated CB MNC 5-day cultures, an effect that could be counteracted by IL-15; comparable effects were observed with APB. Short-term (18-h) experiments revealed that CsA down-regulated K562 cytotoxicity of IL-15-activated (P=0.018) but not resting (P=0.268) purified CB NK cells. IL-15-induced CB NK CD69 expression showed increased CsA sensitivity over APB (P=0.012). CsA down-regulated K562 cell-induced CD54 (P=0.028) but not perforin (P=0.416) expression of IL-15-activated CB NK cells. Target-induced apoptosis of IL-15-activated CB (P=0.043) but not APB (P=0.144) NK cells was decreased by CsA. We have demonstrated differential CsA sensitivity of IL-15-activated CB and APB NK cells. These results may be used to improve the design of IL-15-activated NK cell adoptive immunotherapy in cancer patients receiving CsA post-CB transplantation.

Ex Vivo Expansion of Cord Blood NK Cell Have In Vivo Efficacy Against Leukemia.

Introduction Allogeneic stem cell transplantation has demonstrated the ability to prevent leukemic relapse via an immune-mediated graft-vs.-leukemia effect. Natural killer (NK) cells have been shown to comprise a significant component of this anti-leukemia effect and have been reported to enhance engraftment and reduce graft-vs.-host disease. Ex-vivo expansion of peripheral blood-derived NK cells has been demonstrated. Cord blood (CB) is a promising alternate source of NK cells with enhanced cytokine/antigen-responsiveness, proliferation and increasing availability. We studied expansion conditions for the generation of CB NK cells for clinical use, and evaluated them in vivo in NOD-SCID/IL2Rgnull mice engrafted with human leukemia.Methods Two expansion protocols were compared:1.CD56+ NK cells were isolated and cultured with irradiated CD56− feeder cells in RPMI-1640 supplemented with 100ng/mL IL-2 for 21 days.2.CB mononuclear cells were depleted of CD3+ cells prior to culture.In vivo NK cytolytic activity was measured via chromium release assays on leukemia cell lines (K562, Nalm6) and fresh patient leukemia blasts. Functionality was confirmed in vivo as well by injecting 106 K562 cells or 2.5 ×104 Nalm6 cells into NOD-SCID/IL2Rgnull mice. 2 ×106 CB-expanded NK cells were administered on day +1, and an additional 5×106 CB NK cells were administered on day +7. NK killing of leukemic targets was then confirmed by flow cytometry.Results Feeder cells mediated >30-fold expansion of CB NK cells, generating a nearly pure population that contained 97% CD3−CD56+ cells. Expanded CB NK could lyse >90% of K562 targets at an E:T ratio of 10:1, as well as >40% of patient leukemia blast. In CD3− depleted cultures, similar expansions were observed, however only 55% of these cells were CD3−CD56+ with the remainder being 10% CD3+CD56+ NK/T and 35% CD3+ T lymphocytes. In the NOD-SCID~/IL2Rgnull model, ex vivo expanded CB NK cells demonstrated the ability to reduce the leukemia burden of both AML (K562) and ALL (Nalm6) cells >50%.Conclusions We have demonstrated the feasibility of expanding CB NK ex vivo to clinically-relevant doses with minimal manipulation. These expanded CB NK cells demonstrated cytotoxic activity in vitro and in vivo against a variety of human leukemic cell lines and patient leukemia blasts. These results provide rationale for immunotherapy of leukemia with CB-derived NK cells.

Cord Blood NK Cytotoxic Activity Can Be Selelctively Modulated by Accessory.

Introduction The expansion of cord blood (CB) derived natural killer (NK) cells is a promising cell therapy approach. CB NK cell expansion and activation has proven to be feasible and effective. However, the selectivity of NK killing in an allograft setting raises concern. Accessory cells, in particular dendritic cells and mesenchymal stem cells (MSCs), have been shown to interact with and modulate NK cell proliferation and function. We explored the ability of accessory cells to modulate NK activity in the setting of clinically-relevant CB NK expansion protocols.Methods Human MSCs were isolated from normal bone marrow donors. 5 × 106 CD56-selected NK cells or 5 × 107 unselected or CD3-depleted CB mononuclear cells (MNC) (averaging 10% CD56+ NK cells) were layered on top of the MSCs in the presence of 200u/mL IL-2. CB NK cells were tested for cytolytic activity by 51Cr release assay using MSCs and K562 AML cells. IFN-g secretion was detected by Elispot assay.Results Cord blood-derived NK cells activated by exposure to IL-2 efficiently kill allogeneic MSCs in vitro. A 4 hour chromium release assay revealed that CD56-selected CB NK cells lysed 60% of allogeneic MSCs at E:T ratio 10:1. MSC co-culture with CB NK induced IFN-g secretion as determined by IFN-γ Elispot assay. In contrast, unselected or CD3-depleted CB MNCs with equal numbers of CD56+ NK cells were unable to lyse MSCs despite IL-2 activation. This suggests that CB accessory cells can modulate the activation or cytolytic activity of CB-derived NK cells. To address these two possibilities, we tested the ability of these CB cultures to kill a classic NK target, K562 human myeloid leukemia cells. Surprisingly, in all three CB cultures NK efficiently lysed K562 cells, 95% of lysis at 10:1 E:T ratio by chromium release assay suggesting that these accessory cells did not prevent IL-2 activation of NK cells, but selectively-modulated the cytolytic activity. Thus, preserving anti-leukemia killing while protecting non-malignant MSCs.Conclusions Our data provide evidence for selective modulation of cord blood-derived NK cell cytolytic activity by CB accessory cells. Indeed, activation of cord-derived NK cells in the presence on CD3-depleted accessory cells yields NK cells capable of killing K562 target cells but not allogeneic MSCs. Although the underlying mechanism is unknown, this selective modulation of NK cells may be exploited in the clinical expansion of cord blood NK cells.

KIRs Expression and Cytotoxic Activities of Cord Blood Natural Killer Cells Against Acute Lymphoblastic Leukemia with MLL Gene Rearrangement.

MLL-rearranged ALL is associated with an extremely poor prognosis despite intensive chemotherapy and hematopoietic stem cell transplantation (SCT). We have reported that MLL-rearranged ALL is resistant to death-inducing ligands TRAIL and FasL expressed on cytotoxic T lymphocytes, and therefore T-cell mediating graft-versus-leukemia (GVL) effect is not expected post allogeneic SCT. We recently demonstrated that MLL-rearranged ALL cells were effectively killed by allogeneic NK cells from adult peripheral blood (PB) in a perforin-dependent manner when KIR (killer cell Ig-like receptor) ligand incompatibility exists between ALL cells and NK cells. This KIR ligand incompatibility has been reported to possibly reduce the relapse rate of acute myeloid leukemia post SCT. To pursue the clinical implication in KIR ligand incompatible cord blood transplantation (CBT) for the treatment of MLL-rearranged ALL, we examined the KIRs expression on allogeneic NK cells from umbilical cord blood (CB) by flow cytometry and their in vitro cytotoxic activities against MLL-rearranged ALL cell lines by a standard Cr-release assay at an effector-to-target ratio of 20 to 40. The results were compared between NK cells from adult PB and CB, and between KIR ligand compatible and incompatible NK cells. NK cells from CB were enriched by negative selection and classified into 2 groups based on HLA-C alleles; C1/C1 type (n=5) both alleles belonging to group I (Cw1, Cw3 et. al.) and C1/C2 type (n=4) each of alleles belonging to group I and group II (Cw2, Cw4, et. al.). NK cells from adult PB were similarly classified into C1/C1 type (n=4) and C1/C2 type (n=5). All of the MLL-rearranged ALL cell lines established in our laboratory (n=10) were C1/C1 type, and two cell lines with MLL-ENL (KOPN1, KOCL50) were used as targets. K562 lacking HLA class I expression was used as a positive control target. Although there was no significant difference in the HLA-C group II receptor (KIR2DL1, CD158a) expression between CB- and adult PB-NK cells (17.8±6.3% vs. 26.5±15.2%), the HLA-C group I receptor (KIR2DL2/L3, CD158b) expression on CB-NK cells was significantly lower than on adult PB-NK cells (24.3±10.2% vs. 47.4±19.2%, p=0.009). The CD158b expression showed no difference between C1/C1 and C1/C2 types of CB-NK cells, but it expressed higher on C1/C1 type of adult PB-NK cells than on C1/C2 type (58.9±17.4% vs. 35.9±14.0%. p=0.047), suggesting that the CD158b expression on NK cells increases as getting older particularly in C1/C1 type individuals. In the cytotoxic assay, CB-NK cells irrespective of C1/C1 and C1/C2 types exhibited a lower cytotoxicity against K562 compared with adult PB-NK cells (42.0±19.2% vs. 63.6±9.5%, p=0.009). Of importance, although both C1/C1 and C1/C2 CB-NK cells showed a similar cytotoxicity against K562, C1/C2 CB-NK cells exhibited a significantly higher cytotoxicity against C1/C1 MLL-rearranged ALL cell lines than did C1/C1 CB-NK cells when assessed by a relative cytotoxicity to K562 (KOPN1, 0.84±0.19 vs. 0.47±0.13, p=0.028; KOCL-50, 0.87±0.27 vs. 0.40±0.14, p=0.028), suggesting that a loss of inhibitory signal to CD158a on NK cells from leukemia cells can specifically enhance their alloreactivity. Taken together, MLL-rearranged ALL cells are sensitive to killing by KIR ligand incompatible allogeneic CB-NK cells, and therefore the maximal GVL effect against this leukemia could be expected if the specific CB whose NK cells can exert their alloreactivity is selected for CBT.

Immunologie néonatale et greffe de sang de cordon

The increased susceptibility of human newborns to infections is usually ascribed to the immaturity of the neonatal immune system. The neonatal immune system has never met microbial antigens, and thus the repertoire of its adaptative arm (T and B cells) is entirely pre-immune, or "naïve". However this neonatal pre-immune repertoire is similar to the adult pre-immune repertoire, and cord blood natural killer cells studies show that the innate immunity cells harbor the full killing machinery that characterize mature cells. Moreover, human neonates are able to show an adult-like allogeneic response. Taken together, several lines of evidence suggest that the neonatal immune system, although naïve, is fully mature. However, newborns display phenotypic and functional differences with adults in both adaptative and innate arms. Specific properties may explain these differences, as high number of regulatory T cells, low plasmacytoid dendritic cell response to stimuli and high IL-10 production. These properties are in line with the high susceptibility of newborns to infections and the low incidence of graft-versus-host-disease after cord blood transplantation. To explain these differences, we introduce a new model. Although naive, the neonatal immune system is mature, and these functional differences are due to a message originating from the placenta and aimed at inducing the foetus tolerance to its mother. Full understanding of the involved mechanisms will help to protect the newborn against infections and to improve cord blood transplantation outcome.

Enhanced NK Cell Activation, Cytotoxicity and Ex-Vivo Expansion (EvE) of Cryopreserved Cord Blood (CB) Natural Killer (NK) Cells: Potential Role for CB NK Cells in Adoptive Cellular Immunotherapy (ACI).

CD56+ NK subsets exhibit differential NK receptors (NKR ) such as NCR profiles including killer-Ig-like receptors (KIR), C-lectin (NKG2) and natural cytoxicity receptors (NCR) involved with tumor target recognition (Farag et al Blood, 2002). NK cell activation and NK mediated cytolysis is induced by several NKRs such as NCR (i.e. NKp44, NKp46) and NKG2 surface receptors like NKG2D (Moretta et al, Curr Opinion in Immunol, 2004). Target cell killing by activated NK cells via the granule-dependent pathway is a common mechanism of NK and CTLs and degranulation is followed by the expression of lysosomal-associated membrane protein-1 [LAMP-1] on the cell surface (Penack et al, Leukemia, 2005). CB is limited by the absence of available donor effector cells (NK, CTL, LAK and NKT cells) for infusion after UCBT (Cairo, et al, Transfusion, 2005). We have demonstrated the ability to EvE CB in short-term culture (48 hrs) with IL-2, IL-7, IL-12 and anti-CD3 (ABCY) cryopreserved, thawed, recryopreserved, rethawed and EvE (CTCTE) CB with significant increase in CD3−/16+/56+ bright/dim subsets expressing KIR3DL1, KIR2DL1/S1, KIR2DL2 and CD94/NKG2a (Ayello/Cairo et al BBMT, 2006). In this study, we compared short-term culture (48 hrs) with prolonged cultures (4 to 10 days) on expansion, expression of NCR, NKG2, KIR and cytolytic ability and mechanisms in CTCTE CB. Rethawed nonadherent CB cells were cultured (2–10 days) in serum-free media alone or with anti-CD3 (50 ng/ml), IL-2 (5 ng/ml), IL-7 (10 ng/ml) and IL-12 (10 ng/ml) [ABCY]. NKR expression (CD94, NKG2D, Nkp44 and KIR2DS4), intracellular perforin, granzyme B activity and LAMP-1 receptor (CD107a) expression were determined by flow cytometry. Cytoxicity was measured by europium release assay and tumor targets used were K562, Daudi, neuroblastoma (SHSY5Y) and AML (Kasumi-1) at a 20:1 E:T ratio. C-lectin activating receptor CD94/NKG2D was increased at day 7 vs 2 following ABCY EvE (41.4±0.43 vs 23.7±2.%, p<0.001). Significant increases were seen in activating KIR2DS4 at day 10 vs 2 in ABCY in both CD3−/16+/56+dim and bright subsets (16.9±0.4 vs 2.1±0.2% and 22.3±0.3 vs 0.9± 0.2%, p<0.001, respectively). In contrast, NCR expression in CD3−/16+/56+dim NKp44 subset was significantly decreased at day 10 vs 2 of EvE CB in ABCY (15.2±0.7 vs 27.2±0.7%, p<0.001). Granzyme B expression was increased from day 2 to 10 (25.8± vs 45.1± 1.7%, p<0.001) yet perforin was decreased in EvE CB in ABCY at day 7 vs 2 (68.3±2.19 vs 84.3±1.3%, p<0.001). CD107a expression was significantly increased at day 7 vs 2 in ABCY EvE CB (12.95±1.47 vs 69.34±2.22%, p<0.001). In addition, significant increases in cytolytic activity was demonstrated at day 7 vs 2 of EvE CB cells in ABCY against tumor targets K562 (71.5±±0.81 vs 53.8±3.9%, p<0.001), Daudi (63.9±0.73 vs 31.8±1.8%, p<0.001), SYSY5Y (76.8±6.5 vs 57.5±3.4%, p<0.05) and Kasumi-1 (56.6.5±0.4 vs 38±1.1%, p<0.001). In summary, CB MNC may be thawed at time of CB transplantation, recryopreserved, rethawed at a later date, EvE and activated for up to 10 days to yield significantly increased cytotolytic activity against NHL, AML and neuroblastoma with increased expression of NK KAR KIR2DS4 and granzyme B, LAMP-1 degranulation (NK activation) but decreased NK C-lection CD94/NKG2D, NCR NKp44 and perforin expression.

Characterization of Cord Blood Natural Killer and Lymphokine Activated Killer Lymphocytes Following Ex Vivo Cellular Engineering

Cord blood (CB) natural killer (NK) and lymphokine-activated killer (LAK) cytotoxic cells are poorly characterized but might be used to treat minimal residual and/or recurrent malignant disease. Currently, there is no mechanism to use CB for adoptive cancer cellular immunotherapy after CB transplantation (CBT). Recognizing this as a deficiency, we hypothesized that CB aliquots could be engineered ex vivo for potential donor lymphocyte infusion after CBT. Cryopreserved CB aliquots were thawed, depleted of monocytes, and cultured in serum-free medium alone or serum-free medium with anti-CD3 and interleukins 2, 7, and 12 combined with antibody/cytokines for 48 hours. Immunophenotyping, cytotoxicity, and proliferation were evaluated. A significant expansion of CD3 + was seen, in addition to increases in lymphocyte subsets of CD8 +, CD8 +/CD25 +, and CD3 +/45RO + versus medium alone. A significant enhancement of CD3 proliferation ( P < .001), NK cytotoxicity, NK subset expansion, LAK cytotoxicity, and T-helper 1 subset expansion was also demonstrated. Significant enrichment was seen in NK CD16 +/CD56 +bright, CD16 +/CD56 +dim, CD56 +bright and CD56 +dim/KIR3DL1 +, CD56 +bright and CD56 +dim/KIR2DL1 +, CD56 +bright and CD56 +dim/KIR2DL2 + and CD94 +/NKG2a + subsets. These increases in CB NK subsets were in part secondary to augmentation of cell survival. Further, survival of NOD-SCID mice xenografted with human K562 cells and treated with CB cells expanded with antibody/cytokines was significantly higher than that in animals that received no treatment (phosphate buffered saline) and those that were treated with CB ex vivo expanded in medium alone ( P < .005, respectively). These data suggest that cryopreserved CB cells could be ex vivo engineered for potential use as adoptive cancer cellular immunotherapy for donor lymphocyte infusion after CBT.

Human Congenital Infection With Trypanosoma cruzi Induces Phenotypic and Functional Modifications of Cord Blood NK Cells

We studied the phenotype and activity of cord blood natural killer (NK) cells in newborns congenitally infected with Trypanosoma cruzi. We found that the proportion of CD56(bright) NK cells was significantly decreased in cord blood from these newborns, suggesting they may have been recruited to secondary lymphoid organs. The remaining CD56(bright) NK cells exhibited a defective ability in the production of interferon (IFN)-gamma following in vitro activation with interleukin (IL)-12 + IL-2 or IL-12 + IL-15 cytokines, as compared with NK cells from uninfected newborns. In addition, cord blood NK cells from congenitally infected newborns stimulated with cytokines have a decreased release of granzyme B (GrB) when incubated with K562 target cells. This defect in cytotoxic effector function is associated with a reduced surface expression of activating NK receptors (NKp30, NKp46, and NKG2D) on CD56(dim) NK cells compared with uninfected newborns. These alterations of fetal NK cells from congenitally infected newborns may reflect a down-regulation of the NK cell response after an initial peak of activation and could also be the result of T. cruzi modulating the immune response.

Activation and enhancement of ex vivo (EvE) expanded cryopreserved Cord Blood (CB) Natural Killer (NK) cells, cytolytic potential, and NK receptor (NKR) expression: Potential role for CB NK in Adoptive Cellular Immunotherapy (ACI)

Activation and enhancement of ex vivo (EvE) expanded cryopreserved Cord Blood (CB) Natural Killer (NK) cells, cytolytic potential, and NK receptor (NKR) expression: Potential role for CB NK in Adoptive Cellular Immunotherapy (ACI)

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.

Characterization of Cord Blood Natural Killer Cells: Implications for Transplantation and Neonatal Infections

The role of natural killer (NK) cells in hematopoietic stem cell transplantation and in the control of neonatal infections is not yet clear. Donor-versus-recipient NK cell alloreactivity was found to improve outcome in some settings of hematopoietic stem cell transplantation. We hypothesized that the role of NK cells in cord blood (CB) transplantation and neonatal infections may depend on CB NK cell maturation stage. We therefore analyzed the expression of NK cell differentiation/phenotypic markers in human CB, as well as functional properties of purified CB NK cells. CD8 and CD57 expression was lower in CB than in adult NK cells. However, the expression of other differentiation markers was similar, as was cell surface density of CD56, the percentage of late NK cell precursors, interferon-gamma production, and the proliferative response of purified NK cells to IL-2. Spontaneous cytotoxic activity of purified CB NK cells against NK-sensitive targets was low but reached adult levels after treatment with IL-15. Expression of perforin and granzyme B was higher in CB NK cells (90 versus 58% and 86 versus 69%, respectively). intercellular adhesion molecule-1 and CD161 expression was lower in CB. Surprising, fewer CB NK cells expressed L-selectin, a marker of immature NK cells. Taken together, our results suggest that CB NK cells are phenotypically and functionally mature.

Maternal and neonatal lymphocyte subpopulations at delivery and 3 days postpartum: increased coexpression of CD45 isoforms.

Huge physiologic changes during parturition involve immune cells. Alterations in maternal and neonatal lymphocytes postpartum might ascertain the subpopulations that are most affected and may possibly be of importance in the process. Peripheral blood was taken from 20 healthy women at vaginal delivery and 3 days later, concomitantly with cord and peripheral blood from their newborns. Lymphocyte immunophenotyping was done by three-color flow-cytometry. Maternal T helper cells were decreased and natural killer (NK) cells were significantly increased during labor. Percentage of CD4(+) and percentage and absolute count of CD8(+) cells coexpressing CD45RA and CD45RO antigens were higher than 3 days later. In cord blood NK cells were considerably increased and more CD4(+) cells expressed CD45RO antigen. Coexpression of CD45RA and CD45RO molecules indicates activation of maternal CD4(+) and CD8(+) lymphocytes. NK cells increase suggests their possible association with parturition processes. Lymphocyte subsets in cord blood correspond to maternal subsets to some extent.

Evaluation of cytotoxic function and apoptosis in interleukin (IL)-12/IL-15-treated umbilical cord or adult peripheral blood natural killer cells by a propidium-iodide based flow cytometry.

Both deficient natural killer (NK) cell effector function and increased propensity to apoptosis of neonatal NK cells contribute to the increased susceptibility to infection in the neonates. Interleukin (IL)-12 and IL-15 are two immunoregulatory cytokines known to enhance cytolytic function of neonatal NK cells. The present study aims to simultaneously investigate the effect of IL-12/IL-15 on K562 cytotoxicity as well as NK cells apoptosis of enriched umbilical cord blood (CB) and adult peripheral blood (APB) NK cells, using flow cytometric cytotoxicity assays. The results indicated that (i) prior to cytotoxicity assays, CB NK cells underwent greater degree of spontaneous apoptosis than did APB NK cells; (ii) both IL-12 and IL-15 inhibited the spontaneous apoptosis of CB NK cells, while IL-15 promoted the apoptosis in APB NK cells; (iii) the deficient K562 cytotoxicity of CB NK cells could be enhanced to levels comparable with that of APB NK cells by IL-15; (iv) IL-15 increased the percentages of apoptosis in NK-K562 conjugates in a dose-dependent manner in both CB and APB with a greater effect seen with APB NK cells; (v) target-induced apoptosis was observed with APB NK cells which were further enhanced with IL-15. However, CB NK cells, unstimulated or IL-15-activated, were resistant to K562-induced apoptosis. Thus, the multi-parameter flow cytometry analysis not only demonstrates better for the deficient CB NK function but also provides greater details for cytotoxic mechanisms of NK cells.

19 Maturation stage of cord blood natural killer cells

19 Maturation stage of cord blood natural killer cells

The effect of cord blood sera on CD69 expression

We have reported previously that cord blood (CB) sera inhibit T cell proliferation and cytokine production, and have suggested that the microenvironment that CB T cells come from may be responsible for the reduced function of CB T cells. In the present study we analysed the effect of CB sera on the expression of CD69, the earliest activation marker, on T and natural killer (NK) cells. CD69 expression on adult NK cells was higher than on CB NK cells ( p<0.05). CB and adult blood T cells presented with a similar proportion of CD69 expressing cells. Incubation of peripheral blood mononuclear cells (PBMC) in the CB sera enhanced CD69 expression on alloactivated T cells ( p<0.05). However there was no difference in the CD69 expression within the population of alloactivated adult NK cells. Stimulation of PBMC with phytohaemagglutinin (PHA) in the presence of different sera resulted in higher CD69 expression on T cells incubated with CB sera ( p<0.05), but was similar on NK cells. Our former experiments documented similar effect of CB sera on CD25 expression on mitogen and allostimulated T cells. Thus CB sera seem to have similar influences on the expression of activation markers on stimulated T cells within PBMC, implying that there may be a factor(s) within CB sera affecting lymphocyte responses and activation status.

Study of cord blood natural killer cell suppressor activity

We tested the immunosuppressive effect of cord blood (CB) natural killer (NK) cells using highly purified CB NK cells in mixed lymphocyte cultures (MLC) containing autologous CB T cells as responders. Control cultures were done without NK cells. Our findings revealed that CB NK cells induced a dose-dependent inhibition of T lymphocyte proliferation as evidenced by decreased 3H-thymidine incorporation in MLC. The T cell alloproliferation was significantly decreased in the presence of an NK cell to responder cell ratio of 0.1, 0.2 or 0.4 compared with control cultures done without NK cells (p=0.02, 0.003 and 0.0002, respectively). T lymphocyte inhibition was also achieved using irradiated CB NK cells and still demonstrable on addition of disparate CB NK and T cells to the MLC. In agreement with previous reports, adult blood NK cells inhibited the alloreactive T cells in the MLC using adult T lymphocytes as responders. Compared to control cultures done without NK cells, statistically significant inhibition of 3H-thymidine incorporation in MLC was observed at a ratio of NK cells to responder cells ratio of 0.2 or 0.4 (p=0.02). To investigate the mechanism whereby CB NK cells can interfere with the development of alloreactive T cells in MLC, we measured the tumour necrosis factor-alpha (TNF-alpha) concentrations in MLC supernatants using NK cell-depleted or unseparated CB mononuclear cells (MNC) as responders. The results revealed significantly high levels of TNF-alpha in the absence of NK cells (p=0.007). We conclude that CB NK cells suppress alloreactive T lymphocytes as do their counterparts in adult blood. However, the high NK to T cell ratio in CB could contribute to a more marked suppressive potential compared to that in adult blood. The mechanism of NK-mediated inhibition is likely related to disruption of the TNF-alpha pathway of T-lymphocyte activation.

An angiocentric orbital lesion with an immature natural killer cell immunophenotype

In this report, a unique case of a large unilateral orbital tumor occurring in a 5-year-old white girl is described. The lesion, which was associated with no systemic clinical symptoms, waxed and waned in size over 12 months and eventually spontaneously resolved. Multiple biopsies were performed, which showed an angiocentric and angioinvasive infiltrate composed of a monotonous population of atypical, immature-appearing, large lymphocytes. Extensive molecular and immunophenotypic studies led to the diagnosis of an Epstein-Barr virus-negative angiocentric lymphoproliferative disorder with an immature natural killer (NK) cell immunophenotype (CD16+CD56−), specifically an immunophenotype expressed by normal cord blood NK cells. HUM PATHOL 32:339-342. Copyright © 2001 by W.B. Saunders Company

Fetal natural killer cell function is suppressed.

Previous studies have reported reduced natural killer (NK) cell activity in cord blood (CB) compared with adult blood mononuclear cell populations. Using a non-radioactive killing assay, we have verified these findings suggesting that either the fetal NK cell function is suppressed or that these cells are functionally immature. We have shown that CB NK cells are functional, since activating them with cytokines known to activate adult NK cells [interleukin-2 (IL-2), IL-12 and IL-15] increased activation. However, resting the cells, which enhanced adult NK cell activity (P < 0.01), had no effect on fetal NK cells (P = 0.2). These results suggested that fetal NK cells have the capacity to kill, but this is suppressed in vitro. This hypothesis was strengthened by our observation that eight of nine CB mononuclear cell populations had their NK activity restored by freeze-thawing, whereas four of five adult peripheral blood mononuclear cells had a reduced killing ability on freeze-thawing. Freeze-thawing removes a population of cells that suppresses CB NK cell function. To determine which was the case we performed extensive phenotypic analysis of the CB populations pre- and post-freezing and found that the percentage of the CD3- CD56+ population within CB increased significantly (P < 0.0005 by paired t-test) with freezing, whereas freeze-thawing had no effect on this population within a normal adult peripheral blood mononuclear cell population. Our data suggest that within CB there is a population of cells, as yet undefined, which may be inhibiting NK cell function. This report therefore shows clear differences between NK cells within the adult periphery and in CB, and may lead to a better understanding of events occurring in vivo.