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

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 209-209 ◽  
Author(s):  
Jessica Hochberg ◽  
Brenton Mar ◽  
Janet Ayello ◽  
Nancy Day ◽  
Carmella van de Ven ◽  
...  
Keyword(s):  
Nk Cells ◽  
Peripheral Blood ◽  
Nk Cell ◽  
Ex Vivo ◽  
K562 Cells ◽  
Cellular Immunotherapy ◽  
Wild Type ◽  
Tumor Target ◽  
Adoptive Cellular Immunotherapy ◽  
Membrane Bound

Abstract 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.

Significant Ex-Vivo Expansion and Functional Activation of Cord Blood (CB) Natural Killer (NK) Cells with A Concomittant Significant Decrease in CB T Cells Following Stimulation with Genetically Engineered K562 Cells (K562-mb15-41BBL).

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 499-499
Author(s):  
Jessica Hochberg ◽  
Janet Ayello ◽  
Carmella VandeVen ◽  
Jeremy Gold ◽  
Evan Cairo ◽  
...  
Keyword(s):  
T Cells ◽  
Nk Cells ◽  
Nk Cell ◽  
Ex Vivo ◽  
K562 Cells ◽  
Fold Increase ◽  
Ex Vivo Expansion ◽  
Genetically Engineered ◽  
Cellular Immunotherapy ◽  
Adoptive Cellular Immunotherapy

Abstract Abstract 499 Introduction: CD56+ NK subsets exhibit differential NK receptors (NKR) such as cytotoxicity profiles including killer-Ig-like receptors (KIR), C-lectin (NKG2) and natural cytotoxicity receptors (NCR) involved with tumor target recognition, which, in part, may play a role in adoptive cellular immunotherapy (ACI) for malignancies (Farag et al Blood, 2002). NK cell activation and NK mediated cytolysis is induced by triggering receptors such as NCR (i.e. NKp46), and NKG2 surface receptors like NKG2D (Moretta et al, Curr Opin in Immunol, 2004, Marcenaro et al, Eur J Immunol, 2003). The major limitations of the use of NK cells in ACI include lack of tumor recognition and/or limited numbers of viable and functionally active NK cells (Shereck/Cairo et al. PBC, 2007). To circumvent these limitations, methods to expand and activate PB NK cells by genetic reengineering have been developed (Imai/Campana et al. Blood, 2005). It has been demonstrated that PB NK cells expanded with modified K562 cells expressing membrane bound IL-15 and 4-1BBL (K562-mb15-41BBL; Imai et al Blood, 2005) are significantly increased in number and maintain heterogeneous KIR expression (Fusaki/Campana et al, BJH, 2009) .We have previously reported the ex-vivo expansion, activation and cytolytic activity of CB NK cells with a cocktail of antibody and cytokines (Ayello/Cairo et al, BBMT, 2006; Ayello/Cairo, Exp Hem, 2009, In Press). Objective: In this study, we compared CB NK expansion and activation following stimulation with genetically engineered K562 cells (K562-mb15-41BBL, generously supplied by D.Campana, St Jude's Children's Hospital, Memphis, TN) with wild-type (WT) K562 cells and NK cell characterization expressing inhibiting and activating KIRs, c-lectin, NCRs and NK cytolytic activation. Methods: Following irradiation with 100Gy, K562-mb15-41BBL or WTK562 were incubated at a 1:1 ratio with fresh CB MNCs at 37C, 5% CO2 for 7 days in RPMI-1640+10IU IL-2. NKR expression (KIR2DS4, NKG2D, NKG2A, CD94, KIR3DL1, KIR2DL2, Nkp46) and LAMP-1 (CD107a) receptor expression and NK cell phenotype (CD56 dim and bright subsets) were determined by flow cytometry. Results: On Day 0, NK cells population was 3.9±1.3%. After 7 days in culture, CB NK cells were significantly increased compared to WTK562 and media alone (72±3.9 vs 43±5.9 vs 9±2.4%, p<0.01). This represented a 35-fold or 3374±385% increase of the input NK cell number. This was significantly increased compared to WTK562 (1771±300%, p<0.05). Concomitantly, there was a significant decrease in CB T cells vs WTK562 or media alone (15±2 vs 36±2 vs 51±7%, p<0.001),respectively. There was a significant increase in CD56bright vs CD56dim populations (67 vs 33%, p<0.01) following stimulation with K562-mb15-41BBL. Also, there was a 10-fold increase in CB NK cells expressing KIR3DL1 following stimulation with K562-mb15-41BBL vs WTK562 (p<0.01) and a 5-fold increase in NK KIR2DS4 expression (p<0.05), respectively. There was a significant increase in the expression of NK activation marker, CD107a, compared to WTK562 (51±0.7 vs 32±1.1,p<0.05). There was no change in CB NK cell expression of the c-lectin receptor, CD94/NKG2A and CD94/NKG2D after stimulation with K562-mb15-41BBL. A standard cryopreserved CB unit (25 ml) contains approximately 750×106 MNC. By using the smaller 5-ml aliquot (20%) of a two-aliquot bag (150×106 MNCs × 3.9%=5.8×106 NK cells), this expansion method would hypothetically yield 200×106 CB NK cells after 7 days stimulation with K562-mb15-41BBL. Conclusion: These results suggest that CB MNC can be ex-vivo expanded with K562-mb15-41BBL resulting in specific expansion of CB NK cells with increased NK KIR expression (KIR2DS4 and KIR3DL1) and NK activation (CD107a), along with a significant decrease in CB T cells. This expansion provides a means to enhance specific CB NK cell expansion for possible use for adoptive cellular immunotherapy in the post UCBT setting Disclosures: No relevant conflicts of interest to declare.

Patients’ Peripheral Blood as a Possible Source of Donor-Derived NK Cells for Ex Vivo Expansion and Genetic Modification for Post-Transplant Cellular Immunotherapy In Cord Blood Recipients

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 2094-2094
Author(s):  
Chihaya Imai ◽  
Sakiko Yoshida ◽  
Takayuki Takachi ◽  
Masaru Imamura ◽  
Ryosuke Hosokai ◽  
...  
Keyword(s):  
Cord Blood ◽  
Nk Cells ◽  
Genetic Modification ◽  
Peripheral Blood ◽  
Nk Cell ◽  
Ex Vivo ◽  
K562 Cells ◽  
Cellular Immunotherapy ◽  
Healthy Individuals ◽  
Expansion Range

Abstract Abstract 2094 Haploidentical natural killer (NK) cells can induce and consolidate remission in patients with high-risk acute myeloid leukemia (AML) (Rubnitz et al. J Clin Onc 24: 371, 2010). Recently, significantly reduced relapse rates were observed in AML patients who received killer immunoglobulin-like receptor ligand-mismatched cord blood, suggesting effective alloreactivity of cord blood-derived NK cells (Willemze et al. Leukemia 23: 492, 2009). Cord blood transplantation (CBT) is an effective alternative source for allogeneic hematopoietic cell transplantation in both children and adults. However, its therapeutic efficacy for malignant diseases is limited by the lack of available donor effector cells, such as cytotoxic T lymphocytes, lymphokine-activated killer cells, NK-like T cells and NK cells, for treatment of hematological relapse and posttransplant lymphoproliferative disorder and/or for scheduled posttransplant cellular immunotherapy against refractory diseases. We previously reported a method that induces NK cells to proliferate and reliably allows their genetic modification in healthy individuals and leukemia patients in remission receiving maintenance chemotherapy (Imai et al. Blood 106: 376, 2005). To explore the possibility of using patients’ peripheral blood as a source for posttransplant NK cell therapy, we used our method to expand donor-derived NK cells from peripheral blood of CBT recipients early after engraftment. We also examined whether NK cells can be rendered cytotoxic against original leukemia blasts by transferring an antigen-specific artificial immunoreceptor gene. This study was approved by an institutional ethical committee. Patients received CBT for consolidation of hematological malignancy (n=7), neuroblastoma (n=1) or resolution of refractory EBV-associated hemophagocytic syndrome (n=1) with myeloablative (n=7) or reduced intensity conditioning (RIC) regimens (n=2). The patients were enrolled in the study after engraftment and peripheral blood was obtained after appropriate written consent was obtained. A chimerism study using short tandem repeat assays showed complete donor chimerism in all patients except one who received RIC-CBT. The peripheral blood was obtained at a median of 92 days post-CBT (range: 46–303 days) and subjected to ex vivo activation and expansion using a previously described protocol with slight modifications. Briefly, peripheral blood was coincubated with modified K562 cells expressing membrane-bound IL-15 and 4-1BB ligand (K562-mb15-41BBL) in the presence of low-dose IL-2 (10 U/mL). Most patients were on maintenance immunosuppressive therapy with calcineurin inhibitors with (n=3) or without (n=6) systemic corticosteroids. After 7 days of culture, a median 11.0-fold expansion (range: 5.3–28.9-fold) was observed in all but one patient who had been administered chemotherapy with Mylotarg for relapsed AML a few days before the blood sampling. The expansion rate in the first week was less efficient in CBT recipients than in healthy individuals (>20-fold), probably because of the immunosuppressants administered. However, an additional 2-week culture in the presence of high-dose IL-2 (1000 U/mL) yielded a median 206-fold expansion (range: 101–1381-fold in 21 days). The expanded NK cells exhibited upregulation of activating receptors including NKG2D, NCRp30 and NCRp44, and vigorous cytotoxicity against K562 cells (86.8–97.7% at an E/T ratio of 1:1). The NK cells were susceptible to retroviral genetic modification with the MSCV-IRES-GFP vector (median GFP-positive cells, 52.7%, n=10). Finally, peripheral NK cells from patients with acute lymphoblastic leukemia were expanded and transduced with the chimeric immunoreceptor gene anti-CD19-BB-ζ. The donor-derived NK cells expressed large amounts of anti-CD19 chimeric receptors on their surface and killed original leukemia blasts that were highly resistant to NK cell lysis (e.g. anti-CD19 vs. non-signaling receptor: 69% vs. 0% at an E/T ratio of 1:1). These results suggest that, in CBT recipients, ex vivo expansion and genetic modification of donor-derived NK cells from the patients’ peripheral blood is feasible. Because peripheral blood can be easily and repeatedly obtained, the method described here will allow multiple scheduled infusions. This preliminary study may lead to a novel strategy for posttransplant donor-NK cell therapy in CBT recipients. Disclosures: No relevant conflicts of interest to declare.

Sustained Ex Vivo Expansion of Human Peripheral Blood NK Cells Using Artificial APCs Bearing Membrane-Bound IL-21.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3030-3030
Author(s):  
Cecele J Denman ◽  
Lisa M. Kopp ◽  
Vladimir Senyukov ◽  
Sarah Hagemeister ◽  
Jennifer Johnson ◽  
...  
Keyword(s):  
T Cells ◽  
Nk Cells ◽  
Peripheral Blood ◽  
Cell Expansion ◽  
Nk Cell ◽  
Ex Vivo ◽  
Costimulatory Molecules ◽  
Ex Vivo Expansion ◽  
Cell Bank ◽  
Membrane Bound

Abstract Abstract 3030 Poster Board II-1006 Introduction NK cells have therapeutic potential for a wide variety of human malignancies. The major obstacle for adoptive NK cell immunotherapy is obtaining sufficient cell numbers, as these cells represent a small fraction of peripheral white blood cells, expand poorly ex vivo, and have limited life spans in vivo. Common gamma-chain cytokines are important in NK cell activation, maturation, and proliferation. Others have described improved ex vivo expansion of NK cells using soluble cytokines, when cocultured with stimulated peripheral blood mononuclear cells (PBMC) or Epstein Barr Virus (EBV) lymphoblastioid cell lines, or with artificial antigen presenting cells (aAPC) engineered with costimulatory molecules and/or membrane-bound IL-15 (mIL-15). Expansion of NK cells by these methods has been limited by senescence from telomere shortening. To generate clinical-grade T cells for adoptive transfer, our group developed aAPC derived from K562 retrovirally transduced to express the costimulatory molecules CD86 and CD137L. These aAPC were produced as a master cell bank and further genetically modified to express membrane-bound cytokines. Since IL-21 signals via STAT3, and STAT3 is a known activator of telomerase transcription, we investigated whether NK cell expansion with mIL-21 would provide a sustained proliferative advantage over or in combination with mIL-15. Methods K562 aAPC were retrovirally transduced to express CD64, CD86, CD137L, CD19 (Clone 9), and mIL-15 (Clone 4). These clones were further modified by Sleeping Beauty integration of mIL-21 (Clone 9+IL-21 and Clone 4+IL-21). Freshly isolated PBMC from 5 donors were co-cultured with irradiated K562 aAPC (Clone 4, Clone 4+mIL-21, and Clone 9+mIL-21) at a ratio of 2:1 (aAPC:PBMC) in the presence of 50 IU/ml of rhIL-2. Half of the media was changed every two days and cells were re-stimulated with aAPC every seven days at ratio of 2:1. Cells were counted and phenotyped on day 0, 7, 14, and 21 for CD3, CD16, CD56, NKG2D, KIR (2DL1, 2DL2/3, and 3DL1), and NCR (NKp30, NKp44, NKp46). A preclinical SOP to expand PBMC from a 20 mL blood draw was established and additional donors of known HLA type were expanded with Clone 9+mIL-21 for up to 7 weeks. Cytotoxicity function against K562, 721.221, Raji, and AML targets was measured using the Calcien-AM assay (Invitrogen). Telomere length of expanded and fresh NK cells was measured with the FlouFish assay using the telomere specific FITC conjugated (C3TA2)3 PNA probe. Results By day 14, aAPCs bearing mIL-21 induced greater total cell expansion than those with mIL-15 alone (188, 2900, and 2281-fold for Clone 4, Clone 4+mIL-21, and Clone 9+mIL-21, respectively). However, PBMC cultured without mIL-15 contained far fewer co-expanding T cells. Exponential expansion continued for up to 7 weeks without evidence of senescence when mIL-21 was present, reaching a mean of 91,566-fold expansion of the CD3−CD16/56+ population at 4 weeks. NK cells expanded with mIL-21 had increased expression of KIR and NCR, and expressed very high CD16 and NKG2D levels. These NK cells showed much higher cytotoxicity against all targets than fresh NK cells, retained KIR inhibition, and demonstrated enhanced killing via ADCC. Furthermore, telomere lengths of NK cells expanded with Clone 9+mIL-21 were longer than that of fresh NK cells or those expanded without mIL-21, perhaps explaining the continued expansion without senescence. Thus, NK cell expansion is improved using aAPCs expressing mIL-21 rather than mIL-15. We are currently establishing a GMP-grade working cell bank of Clone 9+mIL-21 for use in clinical trials. Funding: Brenda and Howard Johnson Fund, UT MD Anderson Physician Scientist Program Disclosures No relevant conflicts of interest to declare.

scholarly journals Production of colony-stimulating activity by human natural killer cells: analysis of the conditions that influence the release and detection of colony-stimulating activity

Blood ◽  
1989 ◽  
Vol 74 (1) ◽  
pp. 156-164
Author(s):  
V Pistoia ◽  
S Zupo ◽  
A Corcione ◽  
S Roncella ◽  
L Matera ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Nk Cells ◽  
Natural Killer ◽  
Peripheral Blood ◽  
Nk Cell ◽  
K562 Cells ◽  
Cell Suspensions ◽  
Normal Peripheral Blood ◽  
Colony Stimulating Activity ◽  
Culture Supernatants

Highly purified natural killer (NK) cell suspensions were tested for their capacity to release colony-stimulating activity (CSA) in vitro. NK cell suspensions comprised primarily CD16+ cells and were devoid of CD3+ T cells, CD15+ monocytes, and of B cells. CSA was detected in the NK cell supernatants and sustained the growth of myeloid colonies from both normal peripheral blood and bone marrow. CSA could be in part inhibited by pretreating NK cell culture supernatants with a specific goat anti-granulocyte-macrophage colony-stimulating factor (GM-CSF) antiserum. The inhibition, however, was never complete, a finding that suggests that additional factors were responsible for CSA. Incubation of NK cells with K562 cells (an NK-sensitive target) or with normal bone marrow cells resulted in the appearance of a strong colony- inhibiting activity (CIA) in the culture supernatants. Such CIA was demonstrable in an experimental system where bone marrow or peripheral blood progenitors were induced to form myeloid colonies in the presence of conditioned medium by CSA-producing giant cell tumor (GCT) cells. Stimulation of NK cells with NK-insensitive targets failed to induce CIA production. Neutralizing antitumor necrosis factor (TNF) monoclonal antibodies (MoAbs) were found capable of inhibiting CIA present in the supernatants of NK cells stimulated with K562 cells. Following treatment with anti-TNF antibodies, CSA was again detectable in the same supernatants. This finding indicates that induction of TNF production did not concomitantly switch off CSA production by NK cells. Pretreatment of NK cells with recombinant interleukin-2 (rIL-2) or gamma interferon (r gamma IFN) did not change the amount of CSA released. However, treatment with rIL-2 caused the appearance of a factor in the NK cell supernatants capable of sustaining the formation of colonies of a larger size.

scholarly journals Directed Differentiation of Mobilized Hematopoietic Stem and Progenitor Cells into Functional NK Cells with Enhanced Antitumor Activity

Cells ◽  
2020 ◽  
Vol 9 (4) ◽  
pp. 811
Author(s):  
Pranav Oberoi ◽  
Kathrina Kamenjarin ◽  
Jose Francisco Villena Ossa ◽  
Barbara Uherek ◽  
Halvard Bönig ◽  
...  
Keyword(s):  
Nk Cells ◽  
Progenitor Cells ◽  
Peripheral Blood ◽  
Cell Expansion ◽  
Nk Cell ◽  
Ex Vivo ◽  
Feeder Cells ◽  
Hematopoietic Stem ◽  
Cd34 Cells ◽  
Stem And Progenitor Cells

Obtaining sufficient numbers of functional natural killer (NK) cells is crucial for the success of NK-cell-based adoptive immunotherapies. While expansion from peripheral blood (PB) is the current method of choice, ex vivo generation of NK cells from hematopoietic stem and progenitor cells (HSCs) may constitute an attractive alternative. Thereby, HSCs mobilized into peripheral blood (PB-CD34+) represent a valuable starting material, but the rather poor and donor-dependent differentiation of isolated PB-CD34+ cells into NK cells observed in earlier studies still represents a major hurdle. Here, we report a refined approach based on ex vivo culture of PB-CD34+ cells with optimized cytokine cocktails that reliably generates functionally mature NK cells, as assessed by analyzing NK-cell-associated surface markers and cytotoxicity. To further enhance NK cell expansion, we generated K562 feeder cells co-expressing 4-1BB ligand and membrane-anchored IL-15 and IL-21. Co-culture of PB-derived NK cells and NK cells that were ex-vivo-differentiated from HSCs with these feeder cells dramatically improved NK cell expansion, and fully compensated for donor-to-donor variability observed during only cytokine-based propagation. Our findings suggest mobilized PB-CD34+ cells expanded and differentiated according to this two-step protocol as a promising source for the generation of allogeneic NK cells for adoptive cancer immunotherapy.

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).

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 726-726
Author(s):  
Janet Ayello ◽  
Julia Nemiroff ◽  
Prakash Satwani ◽  
Carmella van de Ven ◽  
Evan Shereck ◽  
...  
Keyword(s):  
Nk Cells ◽  
Cell Activation ◽  
Nk Cell ◽  
Ex Vivo ◽  
Granzyme B ◽  
Cellular Immunotherapy ◽  
Common Mechanism ◽  
Short Term ◽  
Cd107a Expression ◽  
Short Term Culture

Abstract 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&lt;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&lt;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&lt;0.001). Granzyme B expression was increased from day 2 to 10 (25.8± vs 45.1± 1.7%, p&lt;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&lt;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&lt;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&lt;0.001), Daudi (63.9±0.73 vs 31.8±1.8%, p&lt;0.001), SYSY5Y (76.8±6.5 vs 57.5±3.4%, p&lt;0.05) and Kasumi-1 (56.6.5±0.4 vs 38±1.1%, p&lt;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.

Ex Vivo Activated Natural Killer (NK) Cells from Myeloma Patients Kill Autologous Myeloma and Killing Is Enhanced by Elotuzumab

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 3666-3666
Author(s):  
Tarun K. Garg ◽  
Susann Szmania ◽  
Jumei Shi ◽  
Katie Stone ◽  
Amberly Moreno-Bost ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Cell Therapy ◽  
Nk Cells ◽  
Nk Cell ◽  
Ex Vivo ◽  
K562 Cells ◽  
Scid Mice ◽  
Target Cells ◽  
Recent Trial ◽  
Nk Cell Therapy

Abstract Immune-based therapies may improve outcome for multiple myeloma (MM) by eradicating chemo-resistant disease. Our recent trial utilizing IL2 activated, killer immunoglobulin-like receptor-ligand mismatched NK cell transfusions from haplo-identical donors yielded (n) CR in 50% of patients. Unfortunately, after NK cell therapy, 2/10 patients had progressive disease, and the median duration of response for the other 8/10 patients was only 105 days (range 58–593). This may have been due to an insufficient dose of alloreactive NK cells and early rejection. Furthermore, appropriate donors were identified for only 30% of otherwise eligible patients. We therefore investigated whether NK cells from MM patients could be expanded and activated to kill autologous MM. We then examined whether pre-treatment of MM cell targets with elotuzumab, a humanized antibody to the MM tumor antigen CS1, could further enhance NK cell-mediated lysis. PBMC from 5 MM patients were co-cultured for 14 days with irradiated K562 cells transfected with 4-1BBL and membrane bound IL15 in the presence of IL2 (300U/ml) as previously described (Imai et al, Blood2005;106:376–383). The degree of NK cell expansion, NK immunophenotype, and ability to kill MM (4 hour 51Cr release assays) were assessed. To determine the ability of ex vivo expanded NK cells to traffic to bone marrow, activated NK cells were injected into the tail vein of NK cell depleted NOD-SCID mice, which were then sacrificed after 48 hours. Flow cytometry for human CD45, CD3, and CD56 was performed on cells from blood, marrow and spleen. There was an average 64-fold expansion of NK cells (range: 8–200) after 2 weeks of co-culture with K562 transfectants. Expansion of T cells was not observed. The NK cell activating receptor NKG2D, and natural cytotoxicity receptors NKp30, NKp44, and NKp46 were up-regulated following the expansion. Expanded NK cells were able to kill autologous MM (E:T ratio 10:1, average 31%, range 22–41%), whereas resting NK cells did not. Pretreatment of autologous MM cells with elotuzumab increased the activated NK cell-mediated killing by 1.7-fold over target cells pretreated with an isotype control antibody. This level of killing was similar to that of the highly NK kill-sensitive cell line K562 (Figure). Autologous PHA blasts and CD34+ stem cells were not killed. Activated human NK cells were detectable in the bone marrow of NOD-SCID mice 48 hours after injection. Ex vivo activation of NK cells from MM patients with K562 transfectants can induce killing of autologous MM and produce large numbers of NK cells for potential therapy. The addition of elotuzumab to activated NK cell therapy enhances anti-MM effects by ADCC thus invoking an additional NK cell-mediated mechanism of MM killing. Importantly, ex vivo activated NK cells traffic to the bone marrow in mice. Autologous NK cell therapy eliminates the issues related to allo-donor availability and early NK cell rejection, and could provide an option for patients refractory to chemotherapy agents. Figure Figure

Co-Transfusion of Donor NK Cells in Haploidentical Stem Cell Transplantation

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 2907-2907
Author(s):  
Peter J. Lang ◽  
Matthias Pfeiffer ◽  
Heiko-Manuel Teltschik ◽  
Ingo Mueller ◽  
Tobias Feuchtinger ◽  
...  
Keyword(s):  
Nk Cells ◽  
Relapse Rate ◽  
Peripheral Blood ◽  
Nk Cell ◽  
Ex Vivo ◽  
Thymidine Uptake ◽  
Nk Activity ◽  
Specific Lysis ◽  
The Mean

Abstract 39 pedatric patients with acute leukemias (ALL (n=19), AML (n=14) and MDS (n=6)) received T and B cell depleted grafts from full haplotype mismatched related donors. Depletion of the G-CSF stimulated leukapheresis products was carried out with CD3/CD19 coated magnetic microbeads and the CliniMACS device and resulted in a median number of 15.9×106 CD34 (2.5–41) stem cells, 147×106 CD56 NK-cells (9–552) and 413×106 CD14 monocytes (101–1100) per kg body weight. Median numbers of residual T and B cells were 56 000 (10 000–192 000) and 26 000 (2000–149 000) respectively. A reduced intensity regimen (melphalan (140mg/m2), thiotepa (10mg/kg), fludarabine (160mg/m2), OKT3 (0.1mg/kg)) was given in most patients. Co-transfused, HLA mismatched NK cells were traced in peripheral blood of 26 patients starting on day +1 with flow cytometry and appropriate HLA antibodies. Mean numbers of donor derived CD56+ cells/μl were: 3 (day 1), 22 (d 3), 17 (d 7), 75 (d 10), 197 (d 14). Theoretically, the mean absolute number of 4.8×106 co-transfused NK cells should have resulted in a mean number of 2000 cells/μl in peripheral blood of the patients. Comparison of this expected amount with the mean number of NK cells measured within the first week postransplant (25/μl, n=17 data points) showed, that only 1.2% of the cells remained in circulating blood. Thus, the majority of donor NKs did not circulate and probably homed to other compartments (bone marrow, lymph nodes). The number of NK cells cotransfused at day 0 partially influenced the speed of NK cell recovery: patients, who received &gt; 100×106 donor NK cells/kg had significantly higher amounts of circulating cells at day 14 than patients, who received &lt;100×106 donor NKs (240 vs. 140/μl, p&lt;0.05). No significant difference was observed after d 14. Recovery of T cells was not influenced. Graft rejection occurred in 13%. This rate was similar to that of a historical control group (15% in patients who received CD34 positive selected grafts and standard conditioning regimens), although our study patients mainly received an intensity reduced regimen. We conclude, that co-transfused cells facilitated hematopoietic engraftment. Our approach resulted in low TRM (10% at d 365) and in a low relapse rate (20% at 2 years) in patients with microscopical remission (&lt;5% blasts), but was insufficient in patients with active disease (80% relapse rate). We therefore investigated options to increase NK cell activity. Cytotoxicity against K562 cells and thymidine-uptake after PHA stimulation were measured prior and post depletion in 30 procedures. Median specific lysis at E:T ratio = 20:1 was 15% prior and 23% post depletion. Thus, NK activity was not hampered by the procedure. Specific lysis was significantly enhanced by pre-incubation with 1000 U/ml Interleukin (IL) 2 (44%, median) or 2ng/ml IL 12 (40%, median) or 1ng/ml IL 15 ( 53%, median) in vitro. In contrast, thymidine-uptake was reduced from 170 000 to 3000 counts due to profound T-cell-depletion. NK activity was weak against patient derived cryopreserved leukemic blasts without stimulation, but could be significantly increased by cytokine incubation in vitro. Therefore, a pilot study with infusions of IL 15 stimulated NK cells in vivo was started. Up to now, 6 patients received a total of 8 infusions with 12×106 - 150×106 ex vivo stimulated NK cells per kg bw without any side effects. Conclusions: co-transfusion of donor NK cells in haploidentical transplantation is feasible. Only a small portion of cells remained in circulating blood and homing to other organs is likely. NK activity could be increased by cytokines; the use of ex vivo IL 15 stimulated NK cells is currently evaluated. Clinical results suggest antileukemic and graft facilitating effects of donor NK cells.

Immune Function in Follicular Lymphoma (FL): Comparison of Patient with Her Healthy Identical Twin Reveals Enhanced NK Cell Responses in the Patient During Spontaneous Remission [KC1].

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 5012-5012
Author(s):  
Elena Gitelson ◽  
Alexander W. MacFarlane ◽  
Kerry S Campbell ◽  
R. Katherine Alpaugh ◽  
Tahseen I. Al-Saleem ◽  
...  
Keyword(s):  
Nk Cells ◽  
Cell Line ◽  
Peripheral Blood ◽  
Partial Remission ◽  
Nk Cell ◽  
Identical Twin ◽  
Effector Memory ◽  
Target Cells ◽  
Tumor Target ◽  
Memory Phenotype

Abstract Abstract 5012 Identical twins are an excellent model in which to study tumor-specific immune responses (Gitelson et al Br J Haem 2002) as it can be postulated that the immune systems are identical. Spontaneous remissions in FL occur but the immunologic mechanisms remain elusive. We investigated adaptive and innate immunologic phenotypes and responses in a 41-year old patient with untreated grade 1 FL, and her healthy identical twin sister. Patients and methods The patient has had a waxing and waning course of FL over 7 years and currently is in spontaneous partial remission (> 50% reduction of generalized lymphadenopathy) and her peripheral blood was negative for t(14:18) by PCR at the time of this analysis. Immunologic responses of her twin, as well as another set of healthy identical twin sisters, were investigated as controls. We studied peripheral blood using 8-color multiparametric flow cytometry for frequencies and phenotypes of NK and regulatory T cells (Tregs). FOXP3+ cells were further analyzed for naïve, central memory and effector memory phenotype. Activation of NK cells and degranulation in response to tumor target cells as measured by Lysosomal-Associated Membrane Protein 1 (LAMP-1) surface expression were investigated. An erythroblastoid cell line (K562) and an EBV transformed MHC-I deficient lymphoblastoid cell line (721.221) were used as targets. Results NK studies revealed that activated CD69+ NK cells were increased in percentage in FL patient (4.43%) compared to her healthy twin (1.82%), and had increased mean fluorescence intensity (MFI) of 1057 vs 357, respectively. In contrast, almost identical frequencies and MFI of activated NK cells were found in the set of healthy twins (0.55% vs 0.54% and 153 vs 153, respectively). NK cells of FL patient exhibited elevated degranulation compared to the healthy twin in response to stimulation by either target cell: LAMP-1 staining MFI 1907 vs 1395, respectively for 721.221 target cells and 1394 vs 1122, respectively for K562 cells, whereas no difference was detected in non-stimulated NK cells (MFI 536 vs 506, respectively). In addition, Killer Cell Immunoglobulin-like Receptor (KIR) analysis revealed a deficit in the percentage of NK cells staining with an antibody recognizing KIR2DL1/S1 in the patient, but not in her healthy twin (7.0% vs 15.1% of NK cells, respectively), whereas no other differences in KIR receptor expression profiles were found for other KIR or in comparing the set of healthy twins. Analysis of frequencies of circulating Tregs revealed no difference between FL patient and her healthy twin: CD4+CD25+FOXP3+ cells represented 4.92% vs 5.78%, respectively of total CD4 cells and CD8+CD25+FOXP3+ cells represented 1.25% vs 1.37%, respectively of total CD8 cells. Analysis of T cell subsets revealed that Tregs in FL patient and in her twin were mainly of effector memory phenotype: CCR7-/CD45RA-/CD45RO+ (82.6% vs 74.4%, respectively) and central memory phenotype: CCR7+/CD45RA-/CD45RO+ (10.7 vs 14.3%, respectively). Conclusions NK cell studies of FL patient revealed increased numbers of activated CD69+ NK cells which correlated with increased degranulation response to tumor target cells and a deficit in the NK cell repertoire, as noted by the deficiency in NK cells expressing KIR2DL1/S1 in the patient compared to the healthy twin, while no differences in frequencies of circulating Tregs or Treg phenotypes were identified. Although the current results are based upon a single sampling, due to limited availability, the observed potentiation of NK cell activity in the patient with FL suggest a potentially important role of NK cells in tumor control during spontaneous partial remission. Follow-up studies of persisting remission, temporary flare-up or at disease progression will be of interest. Disclosures No relevant conflicts of interest to declare.

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