Early NK Cell Proliferation After Umbilical Cord Blood Transplantation Is Associated With Superior Disease-Free Survival Due To Reduced Leukemia Relapse

Compared to traditional chemotherapy, allogeneic hematopoietic cell transplantation (allo-HCT) has the potential of triggering graft vs. leukemia (GVL) reactions that make this procedure uniquely curative. Despite this, relapse remains the most common cause of treatment failure. Early after allo-HCT the donor immune system reconstitutes within the host and natural killer (NK) cells are the earliest lymphocyte population to recover. Previous studies by us and other investigators have linked rapid lymphocyte recovery and/or high NK cell numbers early after transplant with less relapse. Mechanistically, the reasons for this are unknown. We hypothesized that NK cell proliferation would be associated with allo-HCT outcomes. In a large data set with short-term follow-up we compared stem cell source to NK cell proliferation at Day 28 after transplantation (using Ki67+ staining). In patients undergoing autologous (n=117), sibling (n=57), single umbilical cord blood (sUCB) (n=62) or double umbilical cord blood (dUCB) (n=50) transplantation there were significant differences in the median NK cell proliferation (2.1%, 3.3%, 3.8%, and 19.2%, respectively, p<0.0001). These results suggest that NK cell proliferation is increased when patients are transplanted with stem cell sources that have an increased number of HLA mismatches (dUCB). We next examined factors extrinsic to the NK cells to determine whether differences in the four patient groups were due to these factors. Regulatory T cells (Tregs) are known to negatively regulate the proliferation and activation of a variety of cell types, including NK cells. We utilized the percentage and absolute number of Tregs (defined as CD4+CD25highFoxP3+CD127low) at Day 28 (available for a subset of the above patient samples) and correlated it with the percentage of proliferating NK cells at Day 28 after allogeneic transplant (n=212). The median percentage of Tregs within the lymphocyte fraction was 1.49%. In patients with higher than 1.49% Tregs, the mean NK cell proliferation was 10.8% +/-16.4. In contrast, patients with lower than 1.49% Tregs had a mean NK cell proliferation of 21.2% +/-24.3 (p=0.0004). A nearly identical trend was observed for the absolute numbers of Tregs, suggesting that Tregs may play an extrinsic role in NK cell proliferation after allo-HCT. To further address differences in lymphocyte proliferation and clinical outcomes, we used Ki67 staining to assess T cell (CD4+ and CD8+) and NK cell (CD3-CD56+) proliferation at Day 28 in a different subset of patients undergoing dUCB transplant with acute leukemia or MDS (80%) or other malignant disorders using either myeloablative (42%) or reduced intensity conditioning (58%) and a median of one year follow-up (n=53). There was no association of transplant outcomes with T cell (CD4 or CD8) proliferation. In the NK cell compartment there was a wide range in the percentage of proliferating NK cells (0-86%), with a median of 20%. Patients were segregated into high (>20%) and low (<20%) NK cell proliferating groups and assessed for cytokine levels and transplant outcomes. At Day 28 soluble IL15 levels were not different between high and low NK cell proliferating patients. There was no significant association between NK cell proliferation and the probability or time to neutrophil recovery (p=0.15) or treatment related mortality (p=0.88). Excluding the 14 patients who developed aGVHD prior to day of NK cell assessment (Day 28), the high NK proliferating group had a trend toward a higher cumulative incidence of aGVHD (46% vs. 25%, p=0.17). Using multivariable analysis to control for a variety of patient and disease-associated variables, patients with high NK cell proliferation had significantly better disease-free survival (HR=0.29, 95%CI=0.12-0.71, p=0.01) (Figure 1). Strikingly, patients with high NK cell proliferation were 4-times less likely to relapse (HR=0.24, 95%CI=0.08-0.77, p=0.02). Collectively, these studies show that early NK cell proliferation is associated with superior outcomes after dUCB transplantation, due to reduced relapse, and that this is partly modulated by Tregs. Prospective strategies to increase NK cell proliferation may be of therapeutic benefit to improve transplant outcomes.Figure 1Disease Free Survival for patients undergoing dUCB transplant (n=53) based on low (blue) vs high (red) NK cell proliferation at Day 28 post transplant.Figure 1. Disease Free Survival for patients undergoing dUCB transplant (n=53) based on low (blue) vs high (red) NK cell proliferation at Day 28 post transplant. Disclosures: Wagner: Novartis: Research Funding. Miller:Coronado Biosciences: Scientific Advisory Board Other.

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Adult Umbilical Cord Blood Transplantation Following Non-Myeloablative Conditioning; Impact of Increased Cell Dose and 200cGy TBI on Engraftment and Survival.

Blood ◽

10.1182/blood.v108.11.5399.5399 ◽

2006 ◽

Vol 108 (11) ◽

pp. 5399-5399 ◽

Cited By ~ 1

Author(s):

Mitchell Horwitz ◽

David Rizzieri ◽

Gwynn D. Long ◽

Cristina Gasparetto ◽

Ashley Morris ◽

...

Keyword(s):

Cord Blood ◽

Umbilical Cord ◽

Umbilical Cord Blood ◽

Cord Blood Transplantation ◽

Disease Free Survival ◽

Free Survival ◽

Cell Dose ◽

Blood Transplantation ◽

Disease Free

Abstract Reliable engraftment following transplantation of partially matched umbilical cord blood is dependent upon adequate immunosuppression and myelosuppression. The lowest intensity conditioning regimen that will provide reliable cord blood engraftment in adult patients has not been determined. 26 adult patients with a contraindication for myeloablative marrow conditioning due to advanced age or comorbidities, underwent non-myeloablative umbilical cord blood transplantation for hematologic malignancies. The first 13 patients (Cohort 1) were conditioned with Fludarabine 120mg/m2, Cyclophosphamide 2g/m2 and horse Antithymocyte globulin 90mg/kg. Cyclosporine and Mycophenolate Mofetil was administered for GvHD prophylaxis. The median cell nucleated dose was 2.1 × 107/kg and median follow-up is 60 months (Chao 2004 BBMT 10:569). After protocol revision, 200 cGy total body irradiation was added to the regimen described above. In addition, the minimum nucleated cell dose provided from up to two cord blood units was increased to 3 × 107/kg (Cohort 2). With a median follow-up of 12 months, we now report the outcome of cohort 2. Fourteen patients with AML CR1 (2), CR≥2 (5), CML (1), MDS/AML (1), MDS (4), or Follicular Lymphoma (1) and a median age of 54 (range 21–72) were transplanted. Eight patients required dual cord blood grafts to achieve the minimum cell dose. All grafts were at least 4/6 HLA matches (antigen level class I, allele level class II) with the patient, and with each other (dual cord blood grafts). The median cryopreserved and infused nucleated cell dose was 3.6 × 107/kg and 3.5 × 107/kg respectively. Two patients were not evaluable for engraftment due to early toxic death. Two patients experienced primary graft failure. Acute GvHD (grade II skin) was observed in 2 patients. Limited chronic GvHD developed in 2 patients. Parainfluenza type 3 sinusitis and pneumonitis caused significant morbidity in 5 patients. Day 100 treatment-related mortality occurred in 4 patients (30%) due to; infection (2), hemorrhage (2). Relapse occurred in 5 patients (36%). The estimated one year overall and disease-free survival is 25% and 17%, respectively. T-cell recovery was slow. The median CD4 count/ul for engrafted patients was 44 (range 4–516) and 61(range 2–237) at 3 and 6 months following transplantation, respectively. The median CD8 count/ul was 7 (range 0–359) and 108 (range 0–728) at 3 and 6 months following transplantation, respectively. A comparison of results from the two cohorts is presented in the table. The addition of 200cGy and increasing the cell dose facilitated by dual cord blood transplantation doubled the chance for sustained donor engraftment. Improved engraftment was accompanied by increased treatment-related morbidity and mortality, erasing the potential for improved disease-free survival. Disease relapse, in part due to slow immune recovery resulting in impaired anti-tumor response, was the other major impediment to successful outcome. Techniques focused on enhancing immune recovery would significantly improve outcomes of adult cord blood transplantation. Comparison of Cohort 1 vs Cohort 2 Evaluable Patients Med. Nuc Cell Dose/kg D100 TRM(%) Sustained Donor Engraftment (%)† Disease-Free Survival(%)† †estimated (1yr) *P=0.08 **P=NS Flu / Cy / ATG (Cohort 1) 13 2.1 × 107 15 41 15 Flu/Cy/ATG/200cGy(Cohort 2) 12 3.5 × 107 30 83* 17**

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Influence Of DNA Methyltransferese Inhibitors On The Anti-Leukemic Effect Of Umbilical Cord Blood Derived NK Cells Against Acute Myeloid Leukemia

Blood ◽

10.1182/blood.v122.21.4496.4496 ◽

2013 ◽

Vol 122 (21) ◽

pp. 4496-4496

Author(s):

Harry Dolstra ◽

Jeannette Cany ◽

Anniek B. van der Waart ◽

Marleen Tordoir ◽

Basav Nagaraj Hangalapura ◽

...

Keyword(s):

Cord Blood ◽

Nk Cells ◽

Umbilical Cord ◽

Umbilical Cord Blood ◽

Nk Cell ◽

Ex Vivo ◽

Dnmt Inhibitors ◽

Nsg Mice ◽

Drug Concentrations

Natural killer (NK) cell-based immunotherapy is a promising adjuvant, relatively non-toxic therapy approach for AML. However, further improvement of NK cell-based therapy is needed to increase the clinical effect. In this regard, NK cells generated ex vivo from hematopoietic progenitor cells (HPC) may have significant clinical benefits over enriched NK cells from adult donors, including the ability to choose an appropriate killer-cell immunoglobuline-like receptor (KIR)-ligand or KIR B haplotype alloreactive donor, as well as the capacity to reach high therapeutic dosages. Previously, we reported a GMP-compliant, cytokine/heparin-based culture protocol for the ex vivo generation of highly active NK cells from CD34+ HPC isolated from cryopreserved umbilical cord blood (UCB) units. Expansion in closed, large-scale bioreactors yields a clinically relevant dose of NK cells with high purity and cytolytic activity against AML cells in vitro. Currently, a clinical phase I trial with these HPC-NK cells is ongoing in our hospital. Trafficking studies in NOD/SCID/IL2Rgnull (NSG) mice demonstrated that these HPC-NK cells migrate to the bone marrow (BM) as well as to lymphoid organs where in vivo expansion and maturation can take place. Analysis of the chemokine receptor expression profile of UCB-NK cells matched in vivo findings. Particularly, a firm proportion of UCB-NK cells functionally expressed CXCR4, what could trigger BM homing in response to its ligand CXCL12. In addition, high expression of CXCR3 and CCR6 supported the capacity of UCB-NK cells to migrate to inflamed tissues via the CXCR3/CXCL10-11 and CCR6/CCL20 axis. Importantly, a single HPC-NK cell infusion combined with supportive IL-15 administration was shown to efficiently inhibit growth of K562 leukemia cells implanted in the femur of NSG mice, resulting in significant prolongation of mice survival. Furthermore, we investigated whether modulation by the DNA methyltransferase (DNMT) inhibitors Azacytidine (Aza) and Decitabine (Deci) could further potentiate the antileukemic effect of HPC-NK cells against AML cells. In concordance with previous reports, we observed a dose-dependent effect of Aza and Deci on the growth of the AML cell lines THP1 and KG1a. In subsequent NK cell killing assays, we used clinical relevant low drug concentrations to pre-treat AML cells that did not affect HPC-NK cell viability and cytolytic function. Interestingly, increased killing of pre-treated THP1 and KG1a cells by HPC-NK cells could be observed, which was correlated with an increase in the NKG2D ligand ULBP2, the DNAM-1 ligands CD112 and CD155 as well as TRAIL-R2. Notably, maintenance of low-dose DNMT inhibitors during the KG1a/NK co-culture resulted in pronounced AML growth inhibition. To examine the effect of DNMT inhibitors in vivo, THP1.LucGFP-bearing NSG mice were treated with increasing dose of both agents, which were administered according to current standard protocols applied in humans. Data indicated that treatment with Aza or Deci at dosage equivalent in human to 12.5 and 5 mg/m2 respectively was well tolerated with minimal and/or transient weight loss, and efficiently reduced the progression of THP-1.LucGFP cells in vivo. Currently, we explore whether HPC-NK cells and DNMT inhibitors can work together to combat AML in our xenograft models. These preclinical studies may provide a rationale to investigate the possible additive and/or synergistic anti-AML effects of adoptive HPC-NK cell transfer in combination with these DNMT inhibitors in AML patients. Disclosures: Tordoir: Glycostem Therapeutics: Employment. Spanholtz:Glycostem Therapeutics: Employment.

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Excellent Disease-Free Survival after Double Cord Blood Transplantation Using a Reduced Intensity Chemotherapy Only Conditioning Regimen in a Diverse Adult Population.

Blood ◽

10.1182/blood.v106.11.2048.2048 ◽

2005 ◽

Vol 106 (11) ◽

pp. 2048-2048 ◽

Cited By ~ 4

Author(s):

Karen K. Ballen ◽

Thomas R. Spitzer ◽

Beow Yeap ◽

McAfee Steve ◽

Bimalanghsu R. Dey ◽

...

Keyword(s):

Cord Blood ◽

Aplastic Anemia ◽

Chronic Gvhd ◽

Conditioning Regimen ◽

Disease Free Survival ◽

Free Survival ◽

Cord Blood Unit ◽

Disease Free ◽

Reduced Intensity

Abstract Umbilical cord blood is a useful stem cell source for patients without matched related or unrelated donors. However, single cord blood unit transplantation in adults is associated with high transplant related mortality, mostly due to infection. In this study, we used a reduced intensity conditioning regimen followed by infusion of two partially matched cord blood units. The conditioning regimen was fludarabine 30mg/m2/day x 6 days, melphalan 100mg/m2/day x 1 day, and rabbit antithymocyte globulin 1.5 mg/kg/day x 4 days. Cord blood units were a 4/6 or better HLA match or better with each other and with the patient, and contained a minimum combined pre-freeze cell dose of 3.7 x 107NC/kg. GVHD prophylaxis was cyclosporine and mycophenolate mofetil. Twenty-one patients, 15 males (71%) and 6 females (29%), median age 49 years (range 24–63 years) participated in a Phase I study. The diagnoses were AML (n=8), ALL (n=1), NHL (n=5), CLL (n=2), MDS (n=2), Hodgkins Disease (n=2), and aplastic anemia (n=1). Fifteen percent of patients were non Caucasian. The cell doses infused were a median of 4.0 x107 NC/kg (range 3.0–5.3 x107) and 2.0 X105 CD34+ cells/kg (range 0.6–10.0 x105). Two patients (both with MDS complicating aplastic anemia) experienced primary graft failure, and received successful second cord blood transplants using a different conditioning regimen. Among the remaining 19 patients, the median time to an absolute neutrophil count >500 was 20 days (range 15–34 days). The median time to a platelet count >20,000 unsupported were 41 days (range 21–125 days). One patient experienced a secondary graft failure, and is well following infusion of previously stored autologous cells. 4 patients (21%) experienced Grades II-IV acute GVHD, and only one patient (5%) experienced Grade III GVHD. There were no patients with Grade IV GVHD and no deaths from acute GVHD. Twelve patients were evaluable for chronic GVHD, and 3 patients (25%) had chronic GVHD of which one case was extensive disease. The 100 day transplant related mortality was 14%. The deaths were due to a CNS bleed, Epstein Barr virus lymphoproliferative disorder, and staphylococcal sepsis. Chimerism analysis showed predominance of one cord by Day +100 in 79% of patients evaluable for 100-day follow-up. In 85% of these patients the first cord blood unit infused predominated. One patient has had progressive disease. With a median follow-up of 7 months (range 2–16 months), the overall survival is 79% and the disease-free survival is 64%. The projected one year disease-free survival is 64%. In conclusion, 1) engraftment of adult patients appears to be acceptable using double cord blood products and reduced intensity, non TBI conditioning regimen; 2) the risk of serious acute and chronic GVHD is low, 3) patients with aplastic anemia/MDS may require more intensive immunosuppression to allow engraftment, 4) GVL appears to be preserved despite the low T cell dose.

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Analysis of natural killer (NK) cell activity and adhesion molecules on NK cells from umbilical cord blood

European Journal Of Haematology ◽

10.1034/j.1600-0609.2003.00081.x ◽

2003 ◽

Vol 71 (1) ◽

pp. 29-38 ◽

Cited By ~ 38

Author(s):

Hidehisa Tanaka ◽

Shunro Kai ◽

Masao Yamaguchi ◽

Mahito Misawa ◽

Yoshihiro Fujimori ◽

...

Keyword(s):

Cord Blood ◽

Nk Cells ◽

Natural Killer ◽

Adhesion Molecules ◽

Umbilical Cord ◽

Umbilical Cord Blood ◽

Nk Cell ◽

Cell Activity ◽

Nk Cell Activity

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Transplantation of 2 partially HLA-matched umbilical cord blood units to enhance engraftment in adults with hematologic malignancy

Blood ◽

10.1182/blood-2004-07-2717 ◽

2005 ◽

Vol 105 (3) ◽

pp. 1343-1347 ◽

Cited By ~ 653

Author(s):

Juliet N. Barker ◽

Daniel J. Weisdorf ◽

Todd E. DeFor ◽

Bruce R. Blazar ◽

Philip B. McGlave ◽

...

Keyword(s):

Cord Blood ◽

Umbilical Cord ◽

Umbilical Cord Blood ◽

Hematologic Malignancy ◽

Disease Free Survival ◽

Human Leukocyte ◽

Free Survival ◽

Leukocyte Antigen ◽

Cell Dose ◽

Single Donor

AbstractLimited umbilical cord blood (UCB) cell dose compromises the outcome of adult UCB transplantation. Therefore, to augment graft cell dose, we evaluated the safety of the combined transplantation of 2 partially human leukocyte antigen (HLA)–matched UCB units. Twenty-three patients with high-risk hematologic malignancy (median age, 24 years; range, 13-53 years) received 2 UCB units (median infused dose, 3.5 × 107 nucleated cell [NC]/kg; range, 1.1-6.3 × 107 NC/kg) after myeloablative conditioning. All evaluable patients (n = 21) engrafted at a median of 23 days (range, 15-41 days). At day 21, engraftment was derived from both donors in 24% of patients and a single donor in 76% of patients, with 1 unit predominating in all patients by day 100. Although neither nucleated or CD34+ cell doses nor HLA-match predicted which unit would predominate, the predominating unit had a significantly higher CD3+ dose (P < .01). Incidences of grades II-IV and III-IV acute GVHD were 65% (95% confidence interval [CI], 42%-88%) and 13% (95% CI, 0%-26%), respectively. Disease-free survival was 57% (95% CI, 35%-79%) at 1 year, with 72% (95% CI, 49%-95%) of patients alive if they received transplants while in remission. Therefore, transplantation of 2 partially HLA-matched UCB units is safe, and may overcome the cell-dose barrier that limits the use of UCB in many adults and adolescents.

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Umbilical cord blood-derived ILC1-like cells constitute a novel precursor for mature KIR+NKG2A- NK cells

eLife ◽

10.7554/elife.55232 ◽

2020 ◽

Vol 9 ◽

Author(s):

Sabrina Bianca Bennstein ◽

Sandra Weinhold ◽

Angela Riccarda Manser ◽

Nadine Scherenschlich ◽

Angela Noll ◽

...

Keyword(s):

Cord Blood ◽

Nk Cells ◽

Umbilical Cord ◽

Umbilical Cord Blood ◽

Nk Cell ◽

Functional Characterization ◽

Lymphoid Tissues ◽

The Novel ◽

Effector Cells ◽

Notch Ligands

Despite their identification several years ago, molecular identity and developmental relation between human ILC1 and NK cells, comprising group 1 ILCs, is still elusive. To unravel their connection, thorough transcriptional, epigenetic, and functional characterization was performed from umbilical cord blood (CB). Unexpectedly, ILC1-like cells lacked Tbet expression and failed to produce IFNγ. Moreover, in contrast to previously described ILC1 subsets they could be efficiently differentiated into NK cells. These were characterized by highly diversified KIR repertoires including late stage NKG2A-KIR+ effector cells that are commonly not generated from previously known NK cell progenitor sources. This property was dependent on stroma cell-derived Notch ligands. The frequency of the novel ILC1-like NK cell progenitor (NKP) significantly declined in CB from early to late gestational age. The study supports a model in which circulating fetal ILC1-like NKPs travel to secondary lymphoid tissues to initiate the formation of diversified NK cell repertoires after birth.

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Interleukin-21 Induces the Differentiation of Human Umbilical Cord Blood CD34. −lineage− Cells Into Pseudomature Lytic NK Cells.

Blood ◽

10.1182/blood.v114.22.1476.1476 ◽

2009 ◽

Vol 114 (22) ◽

pp. 1476-1476

Author(s):

Giuseppina Bonanno ◽

Maria Corallo ◽

Annabella Procoli ◽

Andrea Mariotti ◽

Luca Pierelli ◽

...

Keyword(s):

Cell Surface ◽

Cord Blood ◽

Nk Cells ◽

Umbilical Cord ◽

Umbilical Cord Blood ◽

Stromal Cell ◽

Nk Cell ◽

K562 Cells ◽

Gm Csf ◽

Ifn Γ

Abstract Abstract 1476 Poster Board I-499 Background: Umbilical cord blood (UCB) is increasingly used as an alternative source of transplantable CD34+ haematopoietic stem cells (HSC) for neoplastic and non-neoplastic diseases. In addition to CD34-expressing HSC, human UCB contains a rare population of CD34−lineage− cells endowed with the ability to differentiate along the T/NK pathway in response to interleukin (IL)-15 and in the presence of a stromal cell support. IL-21 is a four-helix bundle cytokine released by activated CD4+ T cells and by NKT cells. IL-21 is a crucial regulator of NK cell function, whose influence on IL-15-induced differentiation of CD34−lineage− cells has not been investigated previously. The present study was designed and conducted to address whether IL-21 might replace the stromal cell requirements and foster the IL-15-induced NK differentiation of human UCB CD34−lineage− cells. Methods: CD34−lineage− cells were maintained in liquid culture with 10−6M hydrocortisone, 20 ng/ml Flt3-L and 20 ng/ml SCF, with the addition of 50 ng/ml IL-15 and 20 ng/ml IL-21, either alone or in combination. Cultures were established in the absence of feeder cells or serum supplementation. Cytokine-treated cells were used to evaluate the following parameters: a) cell surface phenotype; b) expression of molecular determinants of lymphoid/NK cell differentiation; c) secretion of IFN-γ, GM-CSF, TNF-α and CCL3/MIP-1α; d) cytolytic activity against NK-sensitive tumour cell targets and e) relative amount of Stat1 (Tyr701), Stat3 (Tyr705) and Stat5 (Tyr694) phosphorylation in response to IL-21. For all the above detailed experiments, control cultures were established with UCB-derived CD34+ HSC. Results: Freshly isolated CD34−lineage− cells stained negatively for stem cell-associated (CD34, CD133) and NK/lymphoid surface antigens (CD7, CD56, CD16, CD3, TCRαβ), and comprised 0.22% on average of UCB mononuclear cells (samples analyzed = 8). CD34−lineage− cells proliferated vigorously in response to IL-15 and IL-21 (average fold expansion at week +4 of culture = 42.5) but not to IL-21 alone, and up-regulated phosphorylated Stat1 and Stat3 proteins, in good agreement with previously published reports on the IL-21-induced activation of Stat signaling. CD34−lineage− cells expanded by IL-21 in combination with IL-15 acquired a peculiar lymphoid morphology with heavy cytoplasmic granules. When compared with CD34-derived NK cells, CD34−lineage− cells emerging from IL-15+IL-21-containing cultures expressed very low levels of CD16 and killer-cell immunoglobulin-like receptor (KIR), but high levels of CD56, NKG2D and IL-21 receptor, consistent with pseudo-mature NK cells. IL-21/IL-15-differentiated cells up-regulated mRNA signals for Bcl-2, GATA-3 and Id2, a master switch required for NK-cell development, and harboured un-rearranged TCRγ genes, suggesting that NK commitment under the experimental conditions here established occurs through a pathway that does not include TCR rearrangement. From a functional standpoint, IL-21/IL-15-treated cells secreted copious amounts of IFN-γ, GM-CSF and CCL3/MIP-1α, and expressed cell surface CD107a upon contact with NK-sensitive tumour targets, a measure of exocytosis of NK secretory granules. Specifically, an average 65±11% of CD56+ NK cells differentiated with IL-15+IL-21 stained positively for CD107a in co-cultures established with NK-sensitive K562 cells. NK cell degranulation occurred at significantly lower levels in co-cultures containing K562 cells and IL-15-differentiated CD34−lineage− cells (mean percentage of CD107a+CD56+ NK cells equal to 35±6 at E:T ratio = 1; p < 0.01 compared with cultures containing IL-15+IL-21-matured NK cells), suggesting that IL-15 and IL-21 exerted synergistic effects on NK activity. Finally, NK cells differentiated from CD34+ HSC with either IL-15 alone or IL-15+IL-21 manifested a similar cytotoxic activity to that of cytokine-differentiated CD34−lineage− cells. Conclusions: This study suggests that considerable numbers of highly pure, lytic CD56+CD16−/+ NK cells for adoptive immunotherapy can be obtained from UCB CD34−lineage− cells using a serum-free, feeder cell-free culture system. The findings highlighted herein also shed some light into the developmental intermediates of NK cells that can be differentiated after the exposure of CD34−lineage− cells to IL-21. Disclosures: No relevant conflicts of interest to declare.

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The Use of Double-Unit Umbilical Cord Blood Transplantation In Adults with Hematologic Disease.

Blood ◽

10.1182/blood.v116.21.4579.4579 ◽

2010 ◽

Vol 116 (21) ◽

pp. 4579-4579

Author(s):

Xiao Ma ◽

Wu Depei ◽

Aining Sun

Keyword(s):

Survival Rate ◽

Cord Blood ◽

Umbilical Cord Blood ◽

Cord Blood Transplantation ◽

Disease Free Survival ◽

Umbilical Cord Blood Transplantation ◽

Hematologic Disease ◽

Free Survival ◽

Blood Transplantation ◽

Disease Free

Abstract Abstract 4579 Objective: In this study, we explored the efficiency and toxicity of 38 cases of double-unit umbilical cord blood transplantation (CBT) in adults with hematologic disease. Methods: The tolerance; transplant related complications; survival rate and disease free survival rate were observed and analyzed. A nonmyeloablative conditioning regimen included cyclophosphamide, fludarabine and 2Gy TBI. Cyclosporine combined mycophenolate mofetil and ATG were used to prevent graft versus host disease (GVHD). Results: All these 38 patients tolerated the therapy well while two patients had graft failure. Severe acute GVHD was presented in 6 patients. Chronic GVHD was occurred in 16 patients. Fatal infection complications were occurred in 5 patients (including CMV idiopathic pneumonia in 2 patients) and 4 patients relapsed after transplantation. Neutrophil engraftment obtained on day +17 and platelet reconstitution occurred on day +42 on median. In the follow-up duration of 17 months on median, the expected 2-year relapse mortality was 17.95%; non-relapse mortality was 25.90%; overall survival was 60.80%, and disease free survival was 52.11%. Compare with 39 cases of haplo-identical HSCT in our department, significant delays in engraftment and lower severe GVHD occurred after CBT. There was no apparent difference between the risk of relapse and DFS in both groups. Conclusion: The use of double-unit CBT after reduced intensive conditioning therapy in adults with hematologic disease is an effective and safe treatment. Fatal infection and relapse are the main reasons of failure. Disclosures: No relevant conflicts of interest to declare.

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Phenotypic and functional heterogeneity of human NK cells developing after umbilical cord blood transplantation: a role for human cytomegalovirus?

Blood ◽

10.1182/blood-2011-08-372003 ◽

2012 ◽

Vol 119 (2) ◽

pp. 399-410 ◽

Cited By ~ 163

Author(s):

Mariella Della Chiesa ◽

Michela Falco ◽

Marina Podestà ◽

Franco Locatelli ◽

Lorenzo Moretta ◽

...

Keyword(s):

Cord Blood ◽

Nk Cells ◽

Umbilical Cord ◽

Umbilical Cord Blood ◽

Nk Cell ◽

Cord Blood Transplantation ◽

Cell Subset ◽

Hematologic Malignancies ◽

Cell Development ◽

Hematopoietic Stem

Abstract Natural killer (NK) cells play a crucial role in early immunity after hematopoietic stem cell transplantation because they are the first lymphocyte subset recovering after the allograft. In this study, we analyzed the development of NK cells after intrabone umbilical cord blood (CB) transplantation in 18 adult patients with hematologic malignancies. Our data indicate that, also in this transplantation setting, NK cells are the first lymphoid population detectable in peripheral blood. However, different patterns of NK-cell development could be identified. Indeed, in a group of patients, a relevant fraction of NK cells expressed a mature phenotype characterized by the KIR+NKG2A− signature 3-6 months after transplantation. In other patients, most NK cells maintained an immature phenotype even after 12 months. A possible role for cytomegalovirus in the promotion of NK-cell development was suggested by the observation that a more rapid NK-cell maturation together with expansion of NKG2C+ NK cells was confined to patients experiencing cytomegalovirus reactivation. In a fraction of these patients, an aberrant and hyporesponsive CD56−CD16+p75/AIRM1− NK-cell subset (mostly KIR+NKG2A−) reminiscent of that described in patients with viremic HIV was detected. Our data support the concept that cytomegalovirus infection may drive NK-cell development after umbilical CB transplantation.

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Development of Optimized Protocol for Generation of NK Cells Expressing Chimeric Antigen Receptors from Hematopoietic Stem Cells for Cancer Immunotherapy

Blood ◽

10.1182/blood.v126.23.2044.2044 ◽

2015 ◽

Vol 126 (23) ◽

pp. 2044-2044

Author(s):

Amie Patel ◽

Laurel Christine Truscott ◽

Satiro N. De Oliveira

Keyword(s):

Stem Cells ◽

Cord Blood ◽

Nk Cells ◽

Umbilical Cord ◽

Umbilical Cord Blood ◽

Large Scale ◽

Nk Cell ◽

Culture Media ◽

Antigen Receptors ◽

Hematopoietic Stem

Abstract Background : Natural Killer (NK) cells are innate immune cells that mediate cytotoxicity against tumor and virus-infected cells, and represent a very promising source for adoptive cellular approaches for cancer immunotherapy. Extensive research has been conducted, including clinical trials, attempting to harness their properties. Gene modification of NK cells can direct their specificity and enhance their function, but the efficiency of gene transfer in mature NK cells is very limited. We have previously published a protocol for generation of human NK cells from gene-modified hematopoietic stem cells (HSC) isolated from umbilical cord blood. Generation of NK cells from HSC provides the opportunity of generation of younger NK cells and expansion of specific gene-modified clones starting from a smaller number of previously isolated and cryopreserved initial cells, with added advantage of generation of multiple batches from the same donor. Chimeric antigen receptors (CAR) are engineered fusion proteins that combine the antigen specificity of antigen-binding moieties of monoclonal antibodies and intracellular activation motifs capable to activate immune cells. Preliminary evidence suggests that NK cells with specificity directed by second-generation CAR may have enhanced cytotoxicity. The goal is to develop a protocol with maximal generation of CAR-expressing NK cells from human HSC for clinical applications. We have evaluated the use of HSC isolated from G-CSF-mobilized apheresed peripheral blood mononuclear cells (PBSC), the elimination of serum in culture media and the elimination of feeder stroma. Significance : Development of a protocol for clinical translation and large-scale good manufacturing practice (GMP) compatibility, maximally generating functional CAR-expressing NK cells. G-CSF mobilized peripheral blood stem cells (PBSC) were used because of availability of larger HSC numbers, increasing safety and efficacy. Changes in culture media based on available literature were evaluated to promote generation of larger number of cells. Human AB serum and serum free media were tested to determine the cell yield for large-scale GMP-compatible protocol. Methods : A third-generation lentiviral vector co-delivering CD19-specific CAR and enhanced green fluorescent protein (EGFP) was used for gene modification of primary human PBSC. Gene-modified cells were then co-cultured with OP9-DL1 stromal cells over 35-40 days in six different culture media conditions for evaluation. Medium "A" was our previously published protocol and consisted of alpha-MEM enriched with 20% of fetal bovine serum and recombinant human cytokines SCF 5ng/mL, Flt3L 5ng/mL, IL-7 5ng/mL, and IL-15 10ng/mL. Medium "B" was AIM V enriched with 10% of human AB serum and cytokines SCF 5ng/mL, Flt3L 5ng/mL, IL-7 20ng/mL, and IL-15 50ng/mL. Medium "C" was similar to medium "B" excluding human AB serum. After 10 days of culture, IL-2 10ng/mL was added to all three media ("plus") creating six different conditions. Flow cytometry was used for detection of EGFP expression and NK cell surface markers. Digital droplet PCR was used for analysis of number of integrated viral copies. Feeder-free culture conditions were developed with the addition of recombinant human IGF-1 100ng/mL to AIM V culture media enriched with SCF, Flt-3 and IL-15. Results : NK cell differentiation was achieved in all conditions. Feeder-free conditions seemed to present mature NK cells earlier (days 25-30) than stromal co-culture (days 35-45), but lower cell yield. As previously reported, PBSC had lower yields of NK differentiation as compared to umbilical cord blood, but higher concentrations of IL-7 and IL-15 rescued the differentiation. Total cell yields were 100-220-fold expansions, with highest counts recovered from conditions with higher IL-7 and IL-15. The removal of serum and addition of IL-2 did not seem to affect differentiation or proliferation. CD56+/CD16+/CD94+ NK cells were present in 10-40% of all CD56+ cells. Conclusions : Large-scale GMP-compatible generation of clinically-relevant numbers of gene-modified NK cells from HSC is feasible. Higher doses of cytokines IL-7 and IL-15 successfully increase the yield of NK cells from PBSC. Absence of serum did not decrease differentiation or proliferation. PBSC showed folds of expansion and NK cell differentiation comparable to those obtained from umbilical cord blood. Disclosures No relevant conflicts of interest to declare.

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