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.

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&lt;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&lt;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&lt;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&lt;0.001), and compared to media alone (5.5% ± 0.4%, p&lt;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.

Assessment of PD-1 expression in human resting and activated natural killer cells and murine tumor models.

2019 ◽  
Vol 37 (8_suppl) ◽  
pp. 36-36
Author(s):  
Sean J. Judge ◽  
Cordelia Dunai ◽  
Ian R. Sturgill ◽  
Kevin M. Stoffel ◽  
William J. Murphy ◽  
...  
Keyword(s):  
Gene Expression ◽  
T Cells ◽  
Nk Cells ◽  
Natural Killer ◽  
Nk Cell ◽  
Ex Vivo ◽  
Fold Increase ◽  
Wild Type ◽  
Immunodeficient Mice

36 Background: Blockade of the PD-1/PD-L1/2 axis has revolutionized cancer therapy. Although reinvigorated PD-1+ T cells are the main effectors in the response to checkpoint blockade, the contribution of Natural Killer (NK) cells to PD-1/PD-L1 inhibition is under debate. While PD-1 has been identified on NK cells, this appears to be restricted to small populations under limited conditions. We sought to evaluate the extent of PD-1 expression in mouse and human resting and activated NK cells. Methods: Human NK cells were isolated from healthy donor PBMCs and cancer patients. Ex vivo activation and proliferation techniques included recombinant human cytokine and feeder line co-culture. Murine NK cells were isolated from splenocytes, and PBMCs from wild type and immunodeficient mice. We assessed NK cell surface markers and intracellular cytokine by flow cytometry, and gene expression by quantitative RT-PCR. Results: Over 21-days of ex vivo expansion, expression of PD-1 or PD-L1 on human NK cells was < 1% at all time points, while TIGIT+ expression increased to > 85%. Conversely, ConA stimulation of T cells increased PD-1 expression with no change in TIGIT expression. QRT-PCR demonstrated absent PD-1 expression in purified NK cells compared to a 5-fold increase in PD-1 gene expression in ConA stimulated PBMCs. PD-1/PD-L1 was also < 1% in the NK92 cell line and < 2.5% in peripheral CD56+CD3- NK cells from patients with soft tissue sarcoma (STS). NK cells from digested freshly resected STS show variable PD-1 ( < 10%) and minimal PD-L1 ( < 1%) expression with a small, but measurable population of intra-tumoral NK cells (1% of immune cells). In vivo mouse studies showed < 5% PD-1+ NK cells in spleen and tumor of CT26 tumor-bearing mice, while PD-L1+ NK cells increased in frequency from spleen (5-35%) to tumor (40-95%) in both wild type BALB/C and SCID mice. Conclusions: In contrast to prior studies, we did not observe a substantial PD-1+ population on human or murine NK cells after multiple activation strategies compared to T cells. Contrary to its application in T cells, our data suggest that PD-1 is not a useful marker for NK cell exhaustion/dysfunction. PD-L1 on NK cells may represent an important link between NK and T cell immunotherapy.

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.

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.

Rhesus Macaque NK Cells Expanded Ex Vivo Undergo Similar Phenotypic and Functional Changes Observed With Expanded Human NK Cells Providing An Excellent Model To Optimize Adoptive NK Cell Transfer

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 2028-2028
Author(s):  
Jan K. Davidson-Moncada ◽  
Richard Childs ◽  
Cynthia E. Dunbar ◽  
Robert Reger ◽  
Ritesh Kotecha ◽  
...  
Keyword(s):  
Nk Cells ◽  
Nk Cell ◽  
Ex Vivo ◽  
K562 Cells ◽  
Culture Conditions ◽  
Ex Vivo Expansion ◽  
Cell Transfer ◽  
Single Positive ◽  
Cd56 Expression

Abstract Although pilot clinical trials have shown adoptive NK cell transfer can result in tumor regression in humans with cancer, additional insight from animal models are needed to optimize NK cell proliferation in vivo as well as to improve their homing and retention in the tumor microenvironment. Mice have provided fundamental insights into NK cell biology, although significant divergence from humans as a consequence of evolution limits their usefulness to optimize adoptive transfer of ex vivo expanded NK cells in humans. Rhesus macaques (RM) express orthologues to most human MHC class I and II genes, and unlike mice, express KIRs and CD56, making them phenotypically and functionally similar to human NK cells. Further, unlike murine NK cells, cultured macaque NK cells can be expanded ex vivo for prolonged periods of time augmenting their cytotoxicity. Although recent studies have provided detailed characterization of freshly isolated RM NK cells, little information exists on the phenotype and function of these cells following ex vivo expansion. We investigated the feasibility of expanding RM NK cells ex vivo using culture conditions similar to those used to expand clinical grade human NK cells and characterized the cytotoxic function of subsets of these cells based on CD16 and CD56 expression. NK cells were enriched from RM PBMCs by either bead immunodepletion of CD3+ cells or by flow sorting) CD3-/CD20-/CD14-/CD16+ single positive (CD16SP) or CD3-/CD20-/CD14-/CD16-/CD56+ single positive (CD56SP) or CD3-/CD20-/CD14-/CD16+/CD56+ double positive (CD16/56DP) NK cells. NK cells were expanded in vitro by co-culture with an irradiated human EBV lymphoblastoid cell line (EBV-LCL) in media containing 500IU/uL of human IL-2. In contrast to humans, where resting NK cells from the peripheral blood are mostly CD16/56DP with smaller SP populations, the majority of NK cells from RM peripheral blood were CD16SP (Figure). Despite this difference, RM NK cells appeared phenotypically similar to human NK cells following ex vivo expansion, and had similar expansion kinetics, typically yielding a 100-fold increase in cells that were 99% CD3- and mostly CD16/56DP or CD56SP with a minor fraction of CD16SP cells. Using NKG2A as a pan NK cell marker as well as expression levels of CD16 and CD56, we evaluated for changes in the cytotoxic function associated with ex vivo expansion in 5 distinct NK cell subsets; CDSP16bright, CD16SPdim, CD56SPbright, CD56SPdim, and CD16/56DP. After 7 days expansion, CD16SP populations contracted substantially in contrast to CD16/56DP and CD56SP populations which increased. After 14 days of expansion, the CD16SP population represented <5% of the expanded NK cells, while the CD16/56DP and the CD56SP populations represented approximately 60% and 40% of NK cells respectively, with distinctions between CD16 and CD56 bright and dim populations being lost (figure). To assess the cytotoxicity of specific subsets of expanded vs fresh NK cells, we evaluated CD107a expression at baseline and following co-culture with K562 cells (2hrs;1:1 ratio). Among fresh NK cells, the highest induction of CD107a was in the CD16SPdim subpopulation. In contrast, following ex vivo expansion, CD107a induction was highest in the dominant CD16/56DP population followed in decreasing order by the CDSP16bright and CD56SPbright populations. To confirm these data, we assessed target killing by expanded NK cells using a flow-based cytotoxicity assay measuring Annexin V and 7AAD expression on K562 cells following co-culture with one of 3 different flow-sorted NK populations; a) CD16SP or b) CD56SP or c) CD16/56DP NK cells. Similar to prior data, we found the dominant CD16/56DP populations to have the greatest cytotoxicity against K562 cells (25%) followed by the CD16SP (17%) and CD56SP (15%) populations. Conclusion These data show RM NK cells can be expanded ex vivo using culture conditions similar to those used to expand clinical grade human NK cells. RM NK cells expanded using EBV-LCL feeder cells are phenotypically similar to expanded human NK cells and based on CD16 and CD56 expression, contain subpopulations that have varying degrees of cytotoxicity against K562 targets. The similarities between expanded RM and human NK cells establish this animal model as having excellent potential to optimize adoptive NK cell transfer in humans. We aim to establish this model and test expansions in vivo. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Potential of the NKG2D/NKG2DL Axis in NK Cell-Mediated Clearance of the HIV-1 Reservoir

2019 ◽  
Vol 20 (18) ◽  
pp. 4490 ◽  
Author(s):  
Maria G. Desimio ◽  
Daniela A. Covino ◽  
Margherita Doria
Keyword(s):  
T Cells ◽  
Nk Cells ◽  
Nk Cell ◽  
Ex Vivo ◽  
Virus Reactivation ◽  
Major Drawback ◽  
Virus Eradication ◽  
Latent Hiv ◽  
Hiv 1

Viral persistency in latently infected CD4+ T cells despite antiretroviral therapy (ART) represents a major drawback in the fight against HIV-1. Efforts to purge latent HIV-1 have been attempted using latency reversing agents (LRAs) that activate expression of the quiescent virus. However, initial trials have shown that immune responses of ART-treated patients are ineffective at clearing LRA-reactivated HIV-1 reservoirs, suggesting that an adjuvant immunotherapy is needed. Here we overview multiple lines of evidence indicating that natural killer (NK) cells have the potential to induce anti-HIV-1 responses relevant for virus eradication. In particular, we focus on the role of the NKG2D activating receptor that crucially enables NK cell-mediated killing of HIV-1-infected cells. We describe recent data indicating that LRAs can synergize with HIV-1 at upregulating ligands for NKG2D (NKG2DLs), hence sensitizing T cells that exit from viral latency for recognition and lysis by NK cells; in addition, we report in vivo and ex vivo data showing the potential benefits and drawbacks that LRAs may have on NKG2D expression and, more in general, on the cytotoxicity of NK cells. Finally, we discuss how the NKG2D/NKG2DLs axis can be exploited for the development of effective HIV-1 eradication strategies combining LRA-induced virus reactivation with recently optimized NK cell-based immunotherapies.

scholarly journals Natural Killer Cell Responses in Hepatocellular Carcinoma: Implications for Novel Immunotherapeutic Approaches

Cancers ◽  
2020 ◽  
Vol 12 (4) ◽  
pp. 926 ◽  
Author(s):  
Stefania Mantovani ◽  
Barbara Oliviero ◽  
Stefania Varchetta ◽  
Dalila Mele ◽  
Mario U. Mondelli
Keyword(s):  
Hepatocellular Carcinoma ◽  
T Cells ◽  
Nk Cells ◽  
Natural Killer ◽  
Immune Escape ◽  
Nk Cell ◽  
Ex Vivo ◽  
Killer Cell ◽  
Current Status ◽  
Tumor Immune Escape

Hepatocellular carcinoma (HCC) still represents a significant complication of chronic liver disease, particularly when cirrhosis ensues. Current treatment options include surgery, loco-regional procedures and chemotherapy, according to specific clinical practice guidelines. Immunotherapy with check-point inhibitors, aimed at rescuing T-cells from exhaustion, has been applied as second-line therapy with limited and variable success. Natural killer (NK) cells are an essential component of innate immunity against cancer and changes in phenotype and function have been described in patients with HCC, who also show perturbations of NK activating receptor/ligand axes. Here we discuss the current status of NK cell treatment of HCC on the basis of existing evidence and ongoing clinical trials on adoptive transfer of autologous or allogeneic NK cells ex vivo or after activation with cytokines such as IL-15 and use of antibodies to target cell-expressed molecules to promote antibody-dependent cellular cytotoxicity (ADCC). To this end, bi-, tri- and tetra-specific killer cell engagers are being devised to improve NK cell recognition of tumor cells, circumventing tumor immune escape and efficiently targeting NK cells to tumors. Moreover, the exciting technique of chimeric antigen receptor (CAR)-engineered NK cells offers unique opportunities to create CAR-NK with multiple specificities along the experience gained with CAR-T cells with potentially less adverse effects.

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