Cord Blood NK Research Articles

Targeting Chronic Lymphocytic Leukemia with Cord Blood NK Cells In NSG Model

AbstractAbstract 2453The incompatibility between donor killer cell immunoglobulin-like receptors (KIRs) and their corresponding ligands has been reported to reduce the risk of relapse after haploidentical and human leukocyte antigen (HLA) mismatched hematopoietic stem cell transplantation in patients with acute myeloid leukemia. We tested this KIR-ligand mismatch hypothesis in the context of allogeneic cord blood NK cells as an adoptive transfer of lymphocytes treating residual chronic lymphocytic leukemia (CLL). As a model for targeting malignant B cells in CLL, we examined allogeneic cord blood NK cell function in NOD scid gamma (NSG) mice, which carry the null interleukin-2 receptor gamma chain mutation, as the mice developed leukemia. Positively selected CD56+ cord blood NK cells were expanded ex vivo with interleukin-2 for 14 days. CLL cells were established in the NSG model by infusion of CLL cells obtained from patients. The leukemia that develops in NSG mice resembles human CLL, with a proliferating CD19+CD23+CD5+ B-cell population detected in the bone marrow, spleen, lymph nodes, and peripheral blood. Subsequently, expanded cord blood NK cells (5 × 106 per mouse) were intravenously infused into NSG-CLL mice. The NK cells that were infused into the CLL mice were typed for HLA and KIR (four main KIRs: KIR2DL2, KIR2DL3, KIR3DL1, and KIR2DL1). The CLL patients' samples that had been used in the NSG models were genotyped for KIR ligands (HLA-C group or HLA-Bw4 group and HLA-A3). In the six pairs of cord blood NK and CLL cells typed, all were HLA mismatched. Five pairs were KIR-ligand mismatched; these mice showed robust NK cell–mediated killing of CLL cells 7 days after NK cell infusion. Of interest, although no KIR-ligand mismatch was seen between the cord blood NK cells and CLL cells in one pair, we still observed NK cell–mediated killing of CLL cells in the mice. In this instance, NK cell–mediated cell killing could have been attributed to possible lower expression of HLA ligands by leukemic cells. Overall survival was significantly improved in CLL-NSG mice that had received cord blood NK cell treatment compared with overall survival in untreated mice (Kaplan-Meier analysis, p < 0.03). These results showed that adoptive transfer of allogeneic cord blood NK cells can be effective in killing CLL, and will be explored in the clinic. Disclosures:Gribben: Roche: Consultancy; Celgene: Consultancy; GSK: Honoraria; Napp: Honoraria.

A Novel Method Of Genetically Engineered K562 Cells To Significantly Expand Cord Blood (CB) NK Cells And Increase CB NK Receptor Expression And NK Cell Activation

A Novel Method Of Genetically Engineered K562 Cells To Significantly Expand Cord Blood (CB) NK Cells And Increase CB NK Receptor Expression And NK Cell Activation

NK Receptor (NKR)-Mediated Signaling Pathways in Cord Blood (CB) CD56dim Versus Peripheral Blood (PB) CD56dim NK Cells: Implication for Immaturity in Cord Blood NK CD56dim Innate Immunity

Background: NK cells are characterized by absent CD3 but expression of CD56dim (90%, cytotoxic) and CD56bright (10%, mediator). NK cells may contribute to the immaturity in cord blood innate and adaptive immunity, and play an important role in the GVL effect post CBT. However, little is known regarding the NKR signaling pathways in CB vs PB CD56dim NK cells and its relationship to the cytotoxic activity. We previously demonstrated the ability to ex-vivo expand CB into NK subsets with profound NK in-vitro and in-vivo cytotoxic activity (Ayello/Cairo BBMT 2006). We further observed that there were 33 and 37 proteins over and under expressed by proteomic expression profiling studies of CB vs PB CD56dim (Shereck/Cairo, ASH 2007; ASPHO 2007; AACR 2007). The differential protein expressions included NKG2A, IP3R type 3, NCR3, MAPKAPK5, Notch 2, PLEK, and NF-X1 using both immunophenotype and proteomic profiling studies.Objectives: To understand the importance of NKR signaling pathways in mediating the differential protein expression and thus in regulating the NK cytotoxic activities in CB vs PB CD56dim, we compared the genomic expression pattern in CB vs PB CD56dim.Methods: For CD56dim isolation, first, NK cells were isolated indirectly by magnetic separation from non-NK cells. Second, the pre-enriched NK cells (CD56+/CD3−) from CB and PB were directly labeled with CD16 (FCGR3) MicroBeads, and the CD56+ CD16+ NK cells (CD56dim) were eluted after removing the column from the magnetic field (Miltenyi). Purity of CD56dim NK cells were then examined by flow cytometry (BD FACScan). For genomic studies, total RNA was isolated and reverse transcribed to cDNA using T7-Oligo (dT) primer. cRNA was Biotin-labeled by in vitro transcription. Fragmented biotin-labeled cRNA was hybridized to GeneChip U133A_2 in GCOS-operated Fluidics Station 450, and then scanned by GeneChip Scanner 3000 (Affymetrix). Data were analyzed using Agilent GeneSpring. Signal intensities were compared using one way ANOVA and Welch Test for statistical analysis.Results: There were 193 and 222 genes over and under expressed at the genomic level between CB vs PB CD56dim NK cells, respectively. CB vs PB CD56dim significantly overexpressed NKG2A (2.14F), CD16b (2.46F), KIR2D (2.13F), NKp44 (NCR2; 2.62F), PBX1 (4.29F), ENPEP (3.93F). There was no significant difference in NKR gene expression of CD16a, CD161, NKG2C, and NKp46 in CB vs PB CD56dim. CB vs PB CD56dim underexpressed the following NK genes: IP3R (1.32F), MAPKAPK5 (1.77F), NCR3 (1.24F), ACACB (3.23F), BBS1 (2.00F).Conclusion: CB vs PB CD56dim overexpressed NKG2A, CD16b, KIR2D, and NKp44 genes compared to only NKG2A was overexpressed at the protein level. These results suggest that NKR protein product levels in CB CD56dim may be directly regulated at the translation level, but not the transcription level. The discrepancy of IP3R, ENPEP, PBX1, and MAPKAPK5 gene expression suggest the involvements of IP3 and calcium ions in NKR signaling pathways. Since the Notch2, PLEK, and NF-X1 gene expression patterns were not increased, the augmented protein levels may result from the regulation of protein translation. The potential regulators of this process may include PBX1, ENPEP, ACACB, and BBS1 though the roles of these regulators need to be defined. We conclude that genomic differences between CB vs PB CD56dim may play an important role in regulating NKR signaling pathway, and thus contribute to disparate cytotoxic activity between CB vs PB and suggest a possible explanation for immaturity of cord blood innate and adaptive immunity.

Phenotypic and Functional Heterogeneity of Natural Killer Cells from Umbilical Cord Blood Mononuclear Cells

Natural killer cells (NK) from umbilical cord blood (CB) play an important role in allogeneic stem cell transplantation and defending infections of newborn. Based on the surface expression of CD56 and CD16 or inhibitory and activatory receptors, NK cells could be subdivided into various subsets with distinct functions. To investigate the biological characterization of NK subsets, the phenotypes and intracellular proteins in freshly isolated CB NK subsets were analyzed at the single cell level by flow cytometry in current study. The production of IFN-γ and cytotoxicity against K562 target cells were also evaluated after stimulation with IL-12. The results showed that NK cells from CB could be divided into four subsets on the basis of CD56 and CD16 expression. Interestingly, CB NK cells expressed CD45RA but not CD45RO molecules that is similar to the naïve T cells. Moreover, CD27, a memory T cell marker, highly expressed on CD56hiCD16− NK cells. The killing-associated molecules, NKG2A, NKG2D, CD95 and the intracellular granzyme B and perforin were heterogeneously expressed among the 4 subsets. Addition of IL-12 into cultures resulted in the induction of IFN-γ expression by CD56hiCD16− and CD56loCD16− subsets and the enhancement of NK cytolytic activity. Taken together, this study elucidated the heterogeneity in phenotypes and biological functions of CB NK cells.

Immunophenotypic and Proteomic Characterization of Cord Blood (CB) CD56dim Versus Peripheral Blood (PB) CD56dim NK Cells.

Background: Neonates may succumb to serious infection due to deficiencies in adaptive cellular immunity, however, little is known regarding the immaturity of CB NK cell innate immunity (Satwani/Cairo et al Biol Neonate 2005). NK cells are characterized by absent CD3 but expression of CD56dim (90%, cytotoxic) and CD56brigh t (10%, mediator) (Shankaran et al Nature 2001). NK subsets' function is based on their repertoire of NK receptors (NKRs) (Moretta et al Annu Rev Immunol 2001). Furthermore, the amount of CB NK cells is significantly low in cryopreserved CB for CBT (Cairo et al Transfusion 2005), however, they may play an important role in the graft vs leukemia (GVL) effect post CBT. We recently demonstrated the ability to expand CB into NK phenotype ex-vivo with profound NK cytotoxic activity (Ayello/Cairo et al BBMT 2006).Objectives: To characterize and compare CB vs. PB NK CD56dim NKR and protein expression.Methods: We positively selected CB vs. PB CD56+ cells using immunomagnetic beads (Miltenyi) and sorted into CD3−/CD56bright and CD3−/CD56dim subsets. NKR expression was evaluated using CD16, CD158a {KIR2DL1}, CD158a,h {KIR2DL1 and KIR2DS1}, CD158b {KIR2DL2}, CD161, NKG2A, NKG2C, NKG2D, Nkp44, NKp46. Additionally, quantitative proteomic analysis was performed using extracts of CB and PB CD56Dim cells using cleavable ICAT labeling followed by multi-dimensional liquid chromatography and tandem mass spectrometry (MS/MS) (Lim et al Lab Invest 2004).Results: CB vs. PB CD56dim showed significant increased expression of NKG2A (81.34 ± 1.51 vs. 54.09 ± 4.56, p < 0.03) and NKG2D (94.08 ± 2.36 vs. 77.43 ± 3.93, p < 0.035). There was no significant difference in NKR expression of CD16, KIR2DL1, KIR2DS1, KIR2DL2, CD161, NKG2C, Nkp44, and NKp46 seen in CB vs. PB CD56dim. CB CD56dim vs. CD56bright had significant increased expression of FcgrIII (91.17 ± 1.95 vs. 28.73 ± 11.23, p < 0.03) and KIR2DL2 (31.88 ± 4.39 vs. 3.08 ± 0.99, p < 0.02). Furthermore, there were 33 and 37 proteins over and under expressed by ≥ 2 fold between CB vs. PB CD56dim NK cells, respectively. CB CD56dim overexpressed functional proteins were 46% binding, 17% catalytic, 15% signaling, 15% transcription, 3% enzyme, 2% structural, and 2% transport. CB CD56dim underexpressed Inositol 1,4,5-triphosphate receptor type 3, mitogen-activated protein kinase 5, and natural cytotoxicity triggering receptor 3 precursor by 8.33, 6.67, and 2.5 fold decrease, respectively. CB CD56dim overexpressed neurogenic locus notch homolog protein 2 precursor, pleckstrin homology domain-containing family A member 1, and transcriptional repressor NF-X1 by 16.67, 7.14, and 5.26 fold increase, respectively.Conclusion: While there are many similiarities in CB vs. PB CD56dim, CB CD56dim appear to be quite mature in development compared to other CB immune cell subsets and in fact have overexpression of a significant number of proteins including NKG2D, NKG2A, and several (33) important functional proteins. These studies suggest that CB CD56dim NK cells are similar in development to PB CD56dim NK cells and may contribute to the GVL effect post UCBT.

NK Cell Increase in Neonates from the Preterm to the Full-Term Period of Gestation

Background: Several studies have reported differences in lymphocyte phenotype in preterm and full-term neonates as compared to children and adults. However, a detailed description of the immunologic cell populations of neonates corresponding to the gestational age is needed. This will be helpful for clinical practice to find the best way to prevent neonatal infections or strengthen the immune system of newborns with some kind of immunodeficiency. Objective: To study maturation of the immune system throughout gestation, describing the variations of the lymphocyte populations in function of the gestational week when born. Methods: We performed a descriptive study in 134 healthy newborns (gestation age 25–42 weeks), quantifying the relative and absolute counts of cell populations in umbilical cord blood obtained during delivery, by a four-color flow cytometry single platform. Results: We first compared the very-preterm (25–30 weeks), preterm (31–36 weeks) and full-term (37–42 weeks) neonates. We found higher absolute counts of all cell populations in the full-term group and lower absolute and relative values of NK cells in the very-preterm group. After that, we analyzed the lymphocyte populations week to week (from week 31 to 41) and found the lowest values of T cells (CD4+ and CD8+) for week 36. However, lower percentages of CD4+ T cells and higher percentages of NK cells were observed in week 38 and 41. Conclusion: We found an increase in cord blood NK cells with gestational age, both in terms of absolute counts and of percentage values. Moreover, the %NK cells showed a pattern opposite to %CD4+ T cells along the studied period.

A Screening Platform for Compounds with Potential Immuno-Regulatory Activities Using Human Cord Blood Mononuclear Cells

A systematic and combinatorial approach was adopted using human umbilical cord blood mononuclear cells (hUCB-MNCs) to screen for potential immuno-regulatory compounds. The hUCB-MNCs contain several types of immunogenic cells, which are a suitable material to mimic the in vivo immuno-response after drug treatment. hUCB-MNCs were treated with various natural products such as quercetin, astaxanthin, caffeic acid, bilobalide, eugenol, rutin and gamma-dodecalactone (gamma-DDL). Phenotypic expression analysis revealed that the subpopulation of CD3(+) T cells, CD56(+) NK cells and CD1a(+) dendritic cells apparently increased after being treated with gamma-DDL for 6 days. The expression of CD56 reached a maximum at 72 h with a dose-dependent relationship. The NK cells activation marker (CD69) also elevated following gamma-DDL treatment. These results demonstrated that the gamma-DDL has immuno-regulatory effects to enhance cord blood NK cells population and bioactivities. Such a high-throughput methodology using hUCB-MNCs may be an effective platform for systematically screening potential immuno-regulatory compounds.

Peripheral Blood (PB) CD56bright and CD56dim Do Not Exhibit Differences in NKR Expression Compared to Their Cord Blood (CB) Counterparts: CB CD56dim May Represent an Intermediary between CB CD56bright and PB CD56dim.

NK cells are characterized by absent CD3 and expression of CD56. CD56 NK cells are classified into CD56dim (90%) that are primarily cytotoxic, and CD56bright that secrete cytokines (Kaplan et al PNAS 1998; Shankaran et al Nature 2001). NK subsets carry out their respective functions based on their repertoire of NK receptors (NKRs), both activating and inhibiting (Moretta et al Annu Rev Immunol 2001). PB CD56dim cells express high concentrations of killer-cell immunoglobulin receptors (KIR) and C-type lectin receptors. PB CD56bright NK cells have increased expression of C-type lectin receptors, but have low expression of the KIRs (Nagler J. Immunol 1989). CD16 (the Fcγ receptor III or FcγRIII) which is responsible for binding to antibody-coated targets and initiating antibody-dependent cellular cytotoxicity (ADCC) has increased expression in CD56dim vs CD56bright NK cells (Leibson et al Immunity 1997). While the characterization and function of PB NK subsets and NKR expression are well described, there is little information regarding CB NK subsets and NKRs. We previously demonstrated a significant increase in CB NK NKR expression in ex-vivo engineered CB MNC following 48 hours of induction with IL-2, IL-7, IL-12, and Anti-CD3 (Ayello/Cairo et al BBMT 2006). In this study, we compared PB vs. CB NK subsets and NKR expression. PB and CB cells were positively selected for CD56+ using CD56 magnetic beads (Miltenyi, Aubrun, CA). CD56+ cells were sorted into CD3−/CD56bright and CD3−/CD56dim subsets by FACS sorter and NK receptor expression (CD16, CD158a {KIR2DL1} CD158a, h {KIR2DL1 and KIR2DS1}, CD158b {KIR2DL2}, CD161, NKG2A, NKG2C, NKG2D, Nkp44, NKp46, Becton Dickinson, Mountainview, CA,) of each subset was analyzed by flow cytometry. CD56+ selection yielded >89% purity (PB-96%, CB-89%). There was no statistical difference (mean ± SEM) in the receptor expression between the CB CD56bright and PB CD56bright and also between the CB CD56dim and PB CD56dim. However, PB CD56bright vs. PB CD56dim had increased expression of NKG2A (93.28 ± 2.83 vs. 66.64 ±7.74, p< 0.015), NKG2C (62.11 ±11.79 vs. 8.08 ± 2.98, p< 0.12), NKp44 (61.52 ±10.35 vs. 3.79 ± 2.32, p< 0.005) and NKp46 (92.53 ±3.31 vs. 64.66 ± 12.34, p< 0.05). PB CD56dim had increased expression of CD16 (93.30 ± 2.71 vs. 61.15 ± 11.79, p< 0.07) compared to PB CD56bright. The CB CD56dim compared to CB CD56bright has increased CD16 expression (85.06 ± 6.75 vs. 40.91 ± 5.74, p< 0.004) only. The PB CD56bright only differed from the CB CD56dim with respect to NKp44 (p< 0.021). The CB CD56bright differed from the PB CD56dim with respect to KIR2DL1 (p< 0.026), CD161 (p < 0.05), NKG2A (p < 0.025), and NKG2C (p < 0.05). In conclusion, there does appear to be a definite difference in NKR expression in PB CD56bright vs. PB CD56dim. However, PB vs. CB CD56dim and PB vs. CB CD56bright did not have any statistically significant NKR expression differences suggesting that the subsets have similar phenotypes in their respective cell source. Based on these results, we postulate that the CB CD56dim subset might reflect an intermediary step in development transitioning from CB CD56bright to PB CD56dim.

Optimal NK Cell Expansion Depends on Accessory Cells, Synergy between Physiologic Concentrations of IL-2 and IL-15, and Umbilical Cord Blood (UCB) NK Cell Precursors Expand Better Than Adult NK Cells.

Allogeneic NK cells may play a therapeutic role in treating patients with AML. We have previously shown that high dose cyclophosphamide (120 mg/kg × 1 day) and fludarabine (125 mg/m2 × 5 days) can clear lymphoid space and induce a surge of endogenous IL-15 to expand haploidentical NK cells obtained from CD3-depleted lymphapheresis products from adult donors. In this initial study, 5 of 19 patients achieved remissions and in vivo NK cell expansion. Limitations of this therapy includeinability of NK cells to expand in most patients,development of PTLD (in one patient) andinadequate disease control.We hypothesized that contaminating T cells could compete for NK cell expansion, that B-cells may contribute to PTLD, and that a 2-step NK cell purification method using CD3 depletion followed by CD56 selection (CliniMacs) may overcome these problems. We tested this in 9 patients with advanced AML. The purified NK cells, activated with 1000 U/ml IL-2 (16–20 hours), were infused 48 hours after the last fludarabine dose. Patients then received subcutaneous IL-2 (10 MU) every other day × 6 doses to expand NK cells in vivo. None of the 9 pts treated on this protocol achieved remission or exhibited evidence of in vivo expansion. Several studies were designed to investigate this unexpected result. First, we found that the more extensive processing resulted in approximately 1/3 the NK cell recovery compared to CD3 depletion alone (38±% viable NK cells vs. 91±2% respectively). In addition, we questioned whether the contaminating B cells and monocytes that were removed in the 2-step depletion strategy had served a critical role in NK cell activation or expansion. Cytotoxicity assays performed against K562 targets showed that the killing was about 3-fold higher with the purified (CD3-CD56+) product compared the CD3-depleted product alone (P=0.001 at E:T of 6.6:1). Proliferation, measured by a 6-day thymidine assay, was higher in proportion to the higher NK cell content. The only difference between the two NK products was their expansion after 14 days of culture, where the CD3-depleted product, with contaminating B-cells and monocytes, gave rise to greater NK cell expansion (14 ±3-fold) compared to the 2-step purified product (4.5±0.9, n=6, P=0.005). If this finding holds true in vivo, the co-infusion of accessory cells may be required for NK cell expansion. We next developed in vitro assays using very low concentrations (0.5 ng/ml) of IL-2 and IL-15 to understand their role in expansion. IL-2 or IL-15 alone induced low proliferation and the combination was synergistic. Lastly, UCB, a rich source of NK cell precursors, was compared to adult NK cells. In a short term proliferation assay, CD56+ NK cells stimulated with IL-2 + IL-15 expanded better from adult donors (61274±12999, n=6) than from UCB (20827± 6959, n=5, P=0.026) but there was no difference after 14 days in expansion culture suggesting that the only difference is in kinetics. However, UCB depleted of T-cells (enriching for NK cell precursors) exhibited higher fold expansion over 14 days under different culture conditions conducive to NK cell progenitors. In conclusion, NK cell expansion in vitro depends on cell source, IL-2 and IL-15 (increased in vivo after lymphoid depleting chemotherapy) as well as accessory cells. The role of these factors to enhance in vivo expansion is under clinical investigation to further exploit the NK cell alloreactivity against AML targets.

The Addition of Interleukin (IL)-15 to ANTI-CD3, IL-2 and FLT-3 Results in Significant Increase in Expansion, Activation, Proliferation and Survival of Cord Blood (CB) NK Cells and Cytotoxic T-Lymphocyte (CTL) Subsets: Potential for Adoptive Cellular Immunotherapy (ACI).

A limitation of CB as a source for ACI is the limited number of immunoeffector T and NK cell subsets. IL-15 is an important lymphokine regulating NK activity, T-cell proliferation and CTL activities. We have previously demonstrated reduced IL-15 mRNA expression and protein production from CB which may in part contribute to the immaturity of CB cellular immunity (Qian/Cairo et al, Blood 1997). NK cells are characterized by NK receptors which exist in both activatory and inhibitory forms (Moretta et al, Ann Rev Immun 2001). We have previously demonstrated the ex vivo expansion, maturation and activation of fresh CB derived T lymphocytes with anti-CD3, IL-2, IL-12 and IL-7 (Robinson/Cairo et al, Exp Hem 2001). For this study, we compared the expansion, proliferation, activation and survival of CB mononuclear cells (MNC) with IL-2, anti-CD3, FLT-3 ± IL-15. CB MNC were isolated by Ficoll density centrifugation and incubated overnight in AIM-V media. The non-adherent MNC fraction was cultured in AIM-V media alone and with IL-2 (5ng/ml), anti-CD3 (50ng/ml), FLT-3 (50ng/ml) and IL-15 (10 &100ng/ml) for 48 hrs. Cell proliferation was determined by the colorimetric assay system using WST-1. Expression of T & NK cell subsets were analyzed by three color flow cytometry using CD3, CD8, CD4, CD25, CD45RO, CD16, CD56, CD158a, CD158b, NKB1, CD94 & NKG2A monoclonal antibodies. Apoptosis markers were determined by measuring Annexin-V & PI by flow cytometry. Cytotoxicity was determined by europium release assay using K562 & Daudi target cells at 20:1 E:T ratio. Expansion of lymphocyte subsets of CD4+/25+, CD8+/25+, CD3+/45RO+ & CD94/NKG2A was significantly increased with IL-2, anti-CD3, FLT-3 + IL-15(100ng/ml) compared to IL-2, anti-CD3, FLT-3 and compared to media (CD4+/25+: 67.2±1.2 vs 39.19±5.3 vs 3.54±0.3%, p<0.01; CD8+/25+: 57.8±8.6 vs 16.94±0.3 vs 0.99±0.4%, p<0.01; CD3+/45RO+: 77.13±1.0 vs 29.96±1.9 vs 6.69±0.3%, p<0.001; CD94/NKG2A: 3.4±0.68 vs 0.29±0.092 vs 0.23±0.09%, p<0.01, respectively). We also noted a significant increase in CD3−/16+/56+ with IL-2, anti-CD3, FLT-3 + IL-15(100ng/ml) compared to media alone (p<0.01). There was no significant increase in expansion of CD3+/16+/56+, CD3−/56+/158a, CD3−/56+/158b or CD3−/56+/NKB1 in various culture conditions described above. IL-2, anti-CD3, FLT-3 + IL-15(100ng/ml) resulted in significant improvement in cell survival compared to media alone (Annexin-V staining) (3.3±0.26 vs 15.51±3.3%, p<0.05). We also noted increase in cell proliferation with IL-2, anti-CD3, FLT-3 + IL-15(100ng/ml) as compared to IL-2, anti-CD3, FLT-3 vs. media alone (1.11±0.09 vs 0.82±0.01 vs 0.69±0.03 OD, p<0.05). We also noted significant increase in cytotoxicity with IL-2, anti-CD3, FLT-3 + IL-15(100ng/ml) as compared to IL-2, anti-CD3, FLT-3 vs media alone (K562–57.81±8.1 vs 17.5±1.4 vs 2.68±0.6%, p<0.01; Daudi-79.8±1.7 vs 23.69±1.5 vs 6.38±0.6, p<0.001) The combination of IL-2 + anti-CD3 + FLT-3 + IL-15 appears to enhance expansion and cytotoxicity of CB CTL and NK subsets in short term culture in part secondary to inhibition of apoptosis. Future studies are warranted to determine if the combination of anti-CD3 + IL-2 + FLT-3 + IL-15 can enhance CB CTL and NK cells for ACI to induce GVHM effects following UCBT.

Dysregulation of Interleukin (IL)-18 Gene Expression and Protein Production in Basal and Activated Cord Blood (CB) vs. Peripheral Blood (PB) Mononuclear Cells and Differential Effects of IL-18 in CB vs. PB T and NK Cell Immunity.

IL-12 and IL-15 are critically important cytokines that regulate the activation of T-cell lymphocyte and NK cell subsets. We have previously demonstrated reduced IL-12 & IL-15 gene and protein expression between activated CB vs. PB mononuclear cells (MNC) (Lee/Cairo, Blood; 1996 & Qian/Cairo, Blood; 1997). IL-18 has been shown to enhance T-cell cytolytic activity & provide antitumor immunity (Osaki et al, J Immunol 1998). In the present study, we compared IL-18 gene expression, protein production and IL-18 mRNA transcript half-life in CB vs. APB MNC and the effects of IL-18 on IFN-γ protein production from CB vs. APB MNC and ex-vivo expansion and activation of fresh CB MNC upon stimulation with IL-12 + IL-2+anti-CD3 ±IL-18. MNC were isolated from CB and APB by Ficoll gradient centrifugation and cultured in RPMI for 2 hours before the addition of staphylococcus aureus enterotoxin (SEB). 24 hours after SEB, the supernatant was measured for IL-18 by ELISA. Furthermore, basal and activated MNC IL-18 mRNA expression was measured by qRT-PCR in APB & CB. APB & CB IL-18 mRNA Actinomycin D half-life studies were performed as previously described (Lee/Cairo, Blood, 1996). IFN-γ production from IL-18 ± IL-12 activated CB vs. APB MNC was measured by ELISA at 48 hours. For ex-vivo expansion, non-adherent CB MNC were cultured for 48 hours with either AIM-V media or AIM-V + IL-2 (5ng/mL) + IL-12 (10ng/mL) + anti-CD3 (50ng/mL) ± escalating doses of IL-18 (1, 10, or 100ng/mL). We demonstrated that the constitutive levels of IL-18 secreted by APB MNC were significantly higher than CB MNC (16.09±6.09 vs. <10 pg/ml, not detectable, p<0.05). SEB dramatically increased IL-18 levels in APB > CB MNC (20hrs) (628.41±34.08 vs. 358.23±9.82 pg/ml, p<0.05). IL-18 mRNA and protein levels were significantly lower in CB compared to APB MNC (2hrs) (0.329 ± 0.131 vs.1.482 ± 0.505, p<0.05). IL-18 mRNA half life was significantly shorter in CB vs. APB MNC (3.281±0.222 vs. 4.967±0.411 min, p<0.05). IL-18 independently or synergistically with IL-12 induced IFN-γ production from both APB and CB MNC (313.67±83.18 vs. 50.00±6.66 pg/ml, p<0.05). Lymphocyte subset expansion of CD8+/25+, CD4+/25+ and CD16+/56+ was significantly increased with IL-12 +IL-2+anti-CD3 and IL-18(10ng/ml) compared to media alone (CD8+/25+: 31±4.9 vs. 0.25±0.8%, p<0.001; CD4+/25+: 61±9 vs. 2.6±0.7%, p<0.001; CD3−/CD16+/56+: 58±11.2 vs. 16.5±5.5%, p<0.001, respectively). Furthermore, there was also significant increase in CD16+/56+ subset cultured in IL-2, IL-12, anti-CD3 vs. IL-2, IL-12, anti-CD3 and IL-18 at 1, 10 and 100ng/ml (16.07±0.87 vs.39.38±9.92 vs.58.27±11.17 vs.66.71±6.25%, p<0.001). There was also significant increase in NK cytotoxicity with IL-2, IL-12, anti-CD3 vs. IL-2, IL-12, anti-CD3 and IL-18 (10ng/ml) (43.1±3.2 vs. 91.5±2.42; p<0.01). These results suggest a significant decrease in IL-18 mRNA expression and protein production in activated CB vs. APB MNC, which was in part secondary to increased degradation of CB IL-18 mRNA. IL-18 in combination with IL-12, IL-2 and anti-CD3 showed enhanced CB NK expansion and cytotoxicity. These results may have important implications in immune reconstitution and graft versus tumor activity following UCBT.

CCR7 Ligands, SLC/6Ckine/Exodus2/TCA4 and CKβ-11/MIP-3β/ELC, Are Chemoattractants for CD56+CD16−NK Cells and Late Stage Lymphoid Progenitors

Two human CC chemokines, SLC/6Ckine/Exodus2/TCA4 and CKβ-11/MIP-3β/ELC, are previously reported as efficacious chemoattractants for T- and B-cells and dendritic cells. SLC and CKβ-11 share only 32% amino acid identity, but are ligands for the same chemokine receptor, CCR7. In this study, we examined chemotactic activity of SLC and CKβ-11 for NK cells and lymphoid progenitors in bone marrow and thymus. It was found that these two CCR7 ligands are chemoattractants for neonatal cord blood and adult peripheral blood NK cells and cell lines. SLC and CKβ-11 preferentially attract the CD56+CD16−NK cell subset over CD56+CD16+NK cells. SLC and CKβ-11 also demonstrate selective chemotactic activity on late stage CD34−CD19+IgM−B-cell progenitors and CD4+and CD8+single-positive thymocytes, but not early stage progenitors. It was noted that SLC is an efficient desensitizer of CKβ-11-dependent NK cell chemotaxis, while CKβ-11 is a weak desensitizer of SLC-dependent chemotaxis. Taken together, these results suggest that SLC and CKβ-11 have the potential to control trafficking of NK cell subsets and late stage lymphoid progenitors in bone marrow and thymus.

954 STIMULATION OF CORD AND ADULT NK CELLS

Cord blood NK cells have decreased activity but are normal in number as identified by antibody B73.1. To determine if they could be stimulated normally, we studied lymphokine and mitogen stimulation of 11 cord and 10 adult overnight cultures Binding (%B) and killing(%K) of target cell K562 in a single cell assay were compared with % lysis(%L) in a 51Cr release assay. Cord %B, was less than adult(p<.05) in unstimulated (UN) cultures but was no different from adult with IL-2 or ct or γ interferon(IFN). Adult cells significantly responded only to IL-2 (p<.05). IL-2 increased the %K. of the bound cells but the cord %K was still less than adult (p<.05). IFN had no effect on %K of adult or cord cells or the %B of adult cells. In the 51Cr release assay, IL-2 and IFN increased both cord and adult %L (p<.05). PHA, ConA or PWM stimulation of endogenous IL-2 production also increased %L (p<.05) but cord activity always remained less than adult (p<.01). There was no synergy with IL-2 and γ IFN or α and γ IFN. Since small increases in %B and %K do not explain large increases in %L, lymphokines may stimulate NK activity by increasing cycling which, is not measured in the single cell assay. While cord %B responds to lymphokines, the defective %K remains. Thus, cord NK cells may be unable to cycle effectively because of deficient killing.