A Novel CD94+ Immature NK Cell Population Based on NKp80 Expression with ILC3-like Features in Human Secondary Lymphoid Tissue

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 751-751
Author(s):  
Steven D Scoville ◽  
Karen Keller ◽  
Bethany Mundy-Bosse ◽  
Michael A. Caligiuri ◽  
Aharon G. Freud
Keyword(s):  
Protein Expression ◽  
Nk Cells ◽  
Cell Population ◽  
Lymphoid Tissue ◽  
Nk Cell ◽  
Ex Vivo ◽  
Differentiation Potential ◽  
Secondary Lymphoid Tissue ◽  
Stage 4 ◽  
Mrna And Protein Expression

Abstract Natural killer (NK) cells are important for their innate ability to identify and kill cancer cells without previous stimulation. However, many of the mechanisms concerning their differentiation from immature precursor cells are still unknown. Elucidation of these pathways are critical to improving targeted therapies that can both activate as well as overcome inhibitory pathways that prevent an effective response. NK cells are thought to develop in secondary lymphoid tissue (SLT) through five stages distinguished by the expression of CD34, CD117, CD94, and CD16. While virtually all NK cells in blood express the pan-NK cell activating receptor, NKp80, we have discovered a distinct Lin CD94+NKp80-CD16- population that is selectively enriched in SLT. The NKp80- population appears to comprise those cells “in transit” between stage 3 (CD117+CD94-) and stage 4 (CD117loCD94+) when evaluating CD94 and CD117 expression among Lin-CD34- cells by flow cytometry. These ex vivo data suggest that this NKp80- population may represent a novel NK cell developmental intermediate (NKDI). SLT-derived NKp80- cells lack cytolytic granules, show minimal cytotoxicity, and produce negligible amounts of interferon-gamma when compared to NKp80+ NK cells in SLT and blood. In addition, NKp80- cells have lower mRNA and protein expression of TBET and EOMES compared to NKp80+ NK, while expressing these factors at higher levels compared to stage 3 cells. Unexpectedly, the NKp80- population also demonstrates Group 3 innate lymphoid cell (ILC3)-like features ex vivo with expression of surface interleukin-1 (IL-1) receptor 1 and CD127 and mRNA and protein expression of the ILC3-associated transcription factors, AHR and RORC. Moreover, following overnight stimulation with IL-1-beta and IL-23, NKp80- cells produce IL-22, albeit at lower concentrations compared to stage 3 cells. Collectively, these data identify NKp80 as a surrogate marker of functional competence during in vivo human NK cell development and provide evidence for the existence of at least two distinct maturation steps within the previously described stage 4 NK cell population in SLT. Since CD94 expression is currently considered to be NK-restricted, these findings raise new questions regarding the developmental relationship between NK cells and ILC3 in humans. In addition, as ILC3s are primarily used to stimulate an anti-microbial defense while NK cells are functional anti-cancer effector cells, defining the differentiation patterns of these cells can have impacts in both of these areas of research. Studies are ongoing to investigate the in vitro differentiation potential(s) of this novel SLT-derived Lin-CD94+NKp80-CD16- population. Disclosures No relevant conflicts of interest to declare.

KIR Ligation During Ex Vivo Expansion Allows Molding Of The KIR Repertoire

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 2288-2288
Author(s):  
Dean A. Lee ◽  
Vladimir V Senyukov ◽  
Jerome R Trembley
Keyword(s):  
Nk Cells ◽  
Cell Population ◽  
Nk Cell ◽  
Conflicts Of Interest ◽  
Ex Vivo ◽  
Cell Populations ◽  
Hematopoietic Stem ◽  
Hla Ligand ◽  
Cell Subpopulations ◽  
Hla Haplotypes

Abstract NK Cell subpopulations express tremendous diversity through polymorphisms, haplotypes, differential expression, and licensing of the Killer Immunoglobulin-like Receptors (KIR). KIR diversity affects both the predisposition to cancer, and the response to therapies such as hematopoietic stem cell transplantation. Clinical trials that take advantage of the anti-cancer properties of NK cells have been limited to choosing donors on the basis of KIR genotypes and/or HLA haplotypes. Moreover, adoptive immunotherapy approaches have been limited by low NK cell doses. The latter hurdle has been recently mitigated by methods for expanding clinical grade NK cells ex vivo. These approaches for growing large numbers of cells now enable investigation into selecting more potent NK cell subsets for increased therapeutic efficacy. We hypothesized that the desired KIR repertoire could be molded through inhibition of undesirable KIR populations by crosslinking with relevant anti-KIR antibodies during expansion with our previously described method, which produces a mean 30,000-fold expansion of NK cells in 3 weeks. First, we determined that maximum inhibition was obtained when anti-KIR antibodies were applied to previously activated NK cells, crosslinked with secondary antibody, and then restimulated for proliferation. Robust reduction of targeted KIR-positive populations could be achieved for each inhibitory KIR (Fig. 1). When pre-activated with anti-KIR2DL1 for one stimulation cycle, NK cells expressing this KIR were decreased by a median of 70.4% ± 19.3%. Similarly, KIR2DL2/3+ NK cells could be reduced by 56% ± 17.5%, and KIR3DL1+ NK cells could be reduced by 53.5% ± 16.3%. When anti-KIR antibodies were combined, similar suppression of multiple-KIR subpopulations was observed. Other NK cell receptors were not significantly affected during targeted KIR inhibition. We then assessed the resulting NK cell populations for degranulation responses to targets with selected HLA as KIR ligands. Inhibition of KIR-expressing subpopulations during expansion resulted in NK cell populations with enhanced degranulation against tumor cells expressing the HLA ligand of the targeted KIR. Importantly, the cytotoxicity of the bulk NK cell population against HLA-negative targets remained. These results indicate that KIR crosslinking during NK cell propagation enables significant reduction in the targeted KIR subpopulations, resulting in an NK cell population with a selective decrease in KIR inhibition. By utilizing antibody-controlled expansion for molding of the KIR repertoire according to patient HLA type, a personalized NK cell product may be produced with enhanced potency, improving NK cell immunotherapy. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Effect of Aging on NK Cell Population and Their Proliferation at Ex Vivo Culture Condition

2018 ◽  
Vol 2018 ◽  
pp. 1-6 ◽  
Author(s):  
Sellamuthu Subbanna Gounder ◽  
Basri Johan Jeet Abdullah ◽  
Nur Ezzati Izyan Binti Mohd Radzuanb ◽  
Farah Dalila Binti Mohd Zain ◽  
Nurhidayah Bt Mohamad Sait ◽  
...  
Keyword(s):  
Nk Cells ◽  
Cell Population ◽  
Cytokine Production ◽  
Nk Cell ◽  
Ex Vivo ◽  
Age Groups ◽  
Proliferation Ability ◽  
Males And Females ◽  
Ex Vivo Culture ◽  
Proliferation Potential

Age-associated changes in natural killer (NK) cell population, phenotype, and functions are directly attributed to the risk of several diseases and infections. It is predicted to be the major cause of the increase in mortality. Based on the surface density of CD56, NK cells are subdivided into two types, such as CD56brightand CD56dimcells, which represent cytokine production and cytotoxicity. In our study, we have examined the age-associated changes in the NK cell population and their subsets at different age groups of males and females (at a range from 41 to 80 years). We found that the total lymphocyte count significantly dropped upon aging in both genders. Although, the level of total immune cells also dropped on aging, and surprisingly the total NK cell population was remarkably increased with the majority of NK cells being CD56dim. Subsequently, we evaluated the proliferation potential of NK cells and our results showed that the NK cell proliferation ability declines with age. Overall, our findings prove that there is an increase in the circulating NK cell population upon aging. However, the proliferation rate upon aging declines when compared to the young age group (<41 yrs).

scholarly journals Stage 3 immature human natural killer cells found in secondary lymphoid tissue constitutively and selectively express the TH17 cytokine interleukin-22

Blood ◽  
2009 ◽  
Vol 113 (17) ◽  
pp. 4008-4010 ◽  
Author(s):  
Tiffany Hughes ◽  
Brian Becknell ◽  
Susan McClory ◽  
Edward Briercheck ◽  
Aharon G. Freud ◽  
...  
Keyword(s):  
Nk Cells ◽  
Natural Killer ◽  
Lymphoid Tissue ◽  
Messenger Rna ◽  
Nk Cell ◽  
Cytolytic Activity ◽  
Small Subset ◽  
Secondary Lymphoid Tissue ◽  
Interleukin 22 ◽  
Th17 Cytokine

Abstract Considerable functional heterogeneity within human natural killer (NK) cells has been revealed through the characterization of distinct NK-cell subsets. Accordingly, a small subset of CD56+NKp44+NK cells, termed NK-22 cells, was recently described within secondary lymphoid tissue (SLT) as IL-22− when resting, with a minor fraction of this population becoming IL-22+ when activated. Here we discover that the vast majority of stage 3 immature NK (iNK) cells in SLT constitutively and selectively express IL-22, a TH17 cytokine important for mucosal immunity, whereas earlier and later stages of NK developmental intermediates do not express IL-22. These iNK cells have a surface phenotype of CD34−CD117+CD161+CD94−, largely lack expression of NKp44 and CD56, and do not produce IFN-γ or possess cytolytic activity. In summary, stage 3 iNK cells are highly enriched for IL-22 and IL-26 messenger RNA, and IL-22 protein production, but do not express IL-17A or IL-17F.

Identification and Ex Vivo Expansion of a Circulating NK Cell Progenitor Population That Leads to Sustained Production of CD56+ NK Cells

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 850-850 ◽  
Author(s):  
Jan K. Davidson-Moncada ◽  
Taylor Harrison Wand ◽  
Robert N. Reger ◽  
Chuanfeng Wu ◽  
Cynthia E. Dunbar ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Therapy ◽  
Nk Cells ◽  
Cell Population ◽  
Nk Cell ◽  
Ex Vivo ◽  
Significant Heterogeneity ◽  
Inhibitory Receptors

Abstract Natural Killer (NK) cells are a component of the innate immune system that can lyse virally infected cells and tumor cells. Although IL-2 activated and ex vivo expanded NK cells are being developed for adoptive cell therapy of cancer, recent data suggests significant heterogeneity exists within these NK cell populations as defined phenotypically, functionally and developmentally. In order to maximize the efficacy of adoptive NK cell therapy, further insights into the ontogeny and behavior of various NK subsets are needed. NK cells have been historically defined as lymphocytes expressing CD56 but lacking CD3. However, we now report the identification of a population of human peripheral blood mononuclear cells that lack expression of the canonical NK markers CD56 and CD16, but otherwise share phenotypic and functional properties with NK cells, which we term "double negative (DN)" killer cells. Besides CD56 and CD16, these cells lack other standard lymphocyte markers including CD3, CD14, CD19, CD20, and CD33, although the majority of these DN cells express surface markers considered to be NK-specific, including CD8α, CD94, CD224, NKp30, CD127, NKp46, NKG2D, and NKG2A. We sorted CD3- CD19- CD14- CD20- human PBMNCs into CD16- CD56Bright, CD16+ CD56dim, CD16+ CD56- and CD16-CD56-(DN) cellular subsets by flow cytometry, then expanded these subsets in vitro by co-culture with irradiated EBV-transformed LCL feeder cells and IL-2 (500IU/mL). DN cells expanded 40-60 fold after 14 days in culture, with 55% of cells retaining the DN phenotype while 40% of cells acquired expression of CD56 but not CD16 (termed CD56SP). Generation of CD56SP cells from DN cells cultured in vitro suggests that circulating DN cells contain NK cell precursors. When DN cells were expanded for 14 days in culture and the residual remaining DN cells were then resorted and re-expanded for an additional 14 days, exponential cellular growth continued. After this addition 14 days of culture, 10-40% of cells became CD56SP NK cells, while the remainder remained DN. Repeated cycles of resorting residual DN cells demonstrated that the DN subset could continually regenerate additional DN cells as well as produce CD56SP NK cells for at least 45 days, suggesting unique proliferative and differentiative properties of this cell population. CD56SP NK cells generated in a 14 day cell culture from DN cells had up-regulation of a variety of cell surface receptors including CD30 (+57%), CD127 (+0.7%), CD161 (+69%), CD57 (+5.7%), CD8α (+40%), CD244 (+99%), NKp30 (+79%), NKp44 (+53%), CD25 (+3.5%), CD69 (+95%), and had higher intracellular IFN-γ (+35%), and perforin (+60%), suggesting both NK differentiation and education occurred in these progeny (figure). In contrast, expression of inhibitory receptors in the DN population, such as CD94 (88%), CD158e (12%) and NKG2A (24%) showed minimal change in expression levels in their progeny DN cells or CD 56SP NK cells at culture-day 14, consistent with prior studies showing inhibitory receptors are less environmentally influenced. CD56SP cells generated from the DN cells also had higher levels of bone marrow and lymph node homing receptors CXCR4 and CCR7 and possessed superior degranulation capacity against K562 cells compared to pre or post expanded DN or pre-expanded CD56SP cells (figure). Further, gene expression analysis comparing DN vs. CD56SP cells at day 0 and DN and CD56SP cells generated from DN cells at day 14, showed DN cells had a gene expression profile with enhanced expression of genes involved in processes such as cell division and replication, while CD56SP cells generated from DN cells had a more cytotoxic or "active phenotype". Conclusion: CD56- 16- DN cells share many characteristics with canonical NK cells, have long-term in vitro differentiation and proliferation capacity, and may represent a cell population that is a self-renewing progenitor for highly activated and cytotoxic CD56SP NK cells. Isolation and expansion of a progenitor population with long-term NK cell repopulating potential in adoptive NK cell immunotherapy trials could offer advantages over trials transferring more differentiated NK cells with limited in vivo proliferative capacity. Disclosures No relevant conflicts of interest to declare.

scholarly journals CD200R1 Distinguishes Uncommitted Precursors from Functionally Mature NK Cells within the Human Tonsil Stage 4A NK Cell Population

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 993-993
Author(s):  
Youssef Youssef ◽  
Ansel P. Nalin ◽  
Jesse Kowalski ◽  
Megan Broughton ◽  
Matthew Lordo ◽  
...  
Keyword(s):  
Nk Cells ◽  
Single Cell ◽  
Cell Population ◽  
Nk Cell ◽  
Ex Vivo ◽  
Cell Subset ◽  
Cell Development ◽  
Lymphoid Cells ◽  
Lymphoid Tissues ◽  
Human Tonsil

Abstract Natural killer (NK) cells are cytotoxic innate lymphoid cells (ILCs) whose development and anti-tumor functions can be critical for the successful treatment and long-term disease-free survival of patients with hematologic malignancies. In humans, NK cells derive from bone marrow resident hematopoietic progenitor cells that traffic to secondary lymphoid tissues (SLTs) where they complete their terminal differentiation and maturation through a series of developmental stages before returning to the blood as mature NK cells. Although major stages of human NK cell development in SLTs have been clearly defined according to the differential surface expression of CD34, CD117, CD94, NKp80, CD16, and CD57 among lineage antigen (Lin) negative lymphocytes, continued investigation has revealed additional phenotypic and functional heterogeneity at each developmental stage. Through extensive ex vivo single-cell RNA sequencing and flow cytometry analyses we have identified two subsets of tonsil-resident Lin -CD34 -CD117 +/-CD94 +NKp80 -CD16 -CD57 - stage 4A NK cells. These two subsets differ in their expression of the inhibitory receptor, CD200R1, which is not expressed by mature NK cells in the peripheral blood from healthy individuals. The majority of stage 4A cells expressed high amounts of surface CD200R1, which correlated with low gene and undetectable protein expression of intracellular cytolytic granules (perforin and granzymes A, B, K, and M), killer immunoglobulin-like receptors (KIRs), and transcription factors required for terminal NK cell maturation (EOMES, T-BET). In addition, upon ex vivo stimulation with phorbol 12-myristate 13-acetate (PMA) and ionomycin, CD200R1 + stage 4A NKDIs did not produce interferon-gamma (IFN-g), a hallmark feature of mature NK cells. In contrast, many CD200R1 - stage 4A cells constitutively expressed perforin, granzymes, EOMES, and/or T-BET; many expressed KIRs; and many produced IFN-g upon ex vivo stimulation. Furthermore, the frequency of KIR + cells among CD200R1 - stage 4A cells was significantly higher than that among autologous tonsil stage 4B NK cells (Lin -CD34 -CD117 +/-CD94 +NKp80 +CD16 -CD57 -) (20.8 ± 1.65 vs. 8.12 ± 1.66; p &lt; 0.01; n = 14), suggesting that as a population CD200R1 - stage 4A cells are potentially out of sequence in terms of the linear NK cell developmental pathway. Based on these ex vivo findings, we hypothesized that CD200R1 + stage 4A cells represent NK cell precursors, whereas the CD200R1 - stage 4A population contains more mature NK cells that lack NKp80, CD16, and CD57. To further address this hypothesis and to determine their ex vivo potentials for NK cell and non-NK ILC differentiation, we cultured CD200R1 + and CD200R1 - stage 4A cells in vitro in the presence of OP9-DL1 stroma and recombinant human IL-7 and IL-15, conditions previously shown to support all human ILC and NK cell subset differentiation. Under these conditions, both stage 4A populations generated NKp80 + NK cells in bulk and single-cell clonal assays, whereas neither population gave rise to ILC2s (CD294 +) which precede stage 4A NK cells in the developmental scheme. However, while the majority of cultures derived from CD200R1 + stage 4A clones contained ILC3s (CD94 -NKp44 +), significantly fewer clones from CD200R1 - stage 4A cells produced ILC3s (7 of 26 CD200R1 - clones vs. 20 of 23 CD200R1 + clones; p = 0.000587). Moreover, none of the CD200R1 - stage 4A-derived clonal cultures that contained KIR + NK cells contained ILC3s, suggesting that the majority of CD200R1 - stage 4A cells are lineage committed NK cells. Collectively, these data further characterize the heterogeneity of the human tonsil stage 4A NK cell population and identify CD200R1 as a marker distinguishing uncommitted precursor cells from a minor population of cells with otherwise mature NK-associated phenotype and function. In light of the role of CD200R1 in regulating lymphocyte functions in the setting of cancer, further research is warranted to determine its potential role(s) in regulating human NK cell development. Disclosures Blachly: KITE: Consultancy, Honoraria; INNATE: Consultancy, Honoraria; AbbVie: Consultancy, Honoraria; AstraZeneca: Consultancy, Honoraria.

scholarly journals Decrease post-transplant relapse using donor-derived expanded NK-cells

Leukemia ◽  
2021 ◽  
Author(s):  
Stefan O. Ciurea ◽  
Piyanuch Kongtim ◽  
Doris Soebbing ◽  
Prashant Trikha ◽  
Gregory Behbehani ◽  
...  
Keyword(s):  
Nk Cells ◽  
Nk Cell ◽  
Ex Vivo ◽  
Disease Free Survival ◽  
Dependent Manner ◽  
Post Transplant ◽  
Haploidentical Transplantation ◽  
High Doses ◽  
High Level

AbstractIn this phase I/II clinical trial, we investigated the safety and efficacy of high doses of mb-IL21 ex vivo expanded donor-derived NK cells to decrease relapse in 25 patients with myeloid malignancies receiving haploidentical stem-cell transplantation (HSCT). Three doses of donor NK cells (1 × 105–1 × 108 cells/kg/dose) were administered on days −2, +7, and +28. Results were compared with an independent contemporaneously treated case-matched cohort of 160 patients from the CIBMTR database.After a median follow-up of 24 months, the 2-year relapse rate was 4% vs. 38% (p = 0.014), and disease-free survival (DFS) was 66% vs. 44% (p = 0.1) in the cases and controls, respectively. Only one relapse occurred in the study group, in a patient with the high level of donor-specific anti-HLA antibodies (DSA) presented before transplantation. The 2-year relapse and DFS in patients without DSA was 0% vs. 40% and 72% vs. 44%, respectively with HR for DFS in controls of 2.64 (p = 0.029). NK cells in recipient blood were increased at day +30 in a dose-dependent manner compared with historical controls, and had a proliferating, mature, highly cytotoxic, NKG2C+/KIR+ phenotype.Administration of donor-derived expanded NK cells after haploidentical transplantation was safe, associated with NK cell-dominant immune reconstitution early post-transplant, preserved T-cell reconstitution, and improved relapse and DFS. TRIAL REGISTRATION: NCT01904136 (https://clinicaltrials.gov/ct2/show/NCT01904136).

scholarly journals Role and Modulation of NK Cells in Multiple Myeloma

Hemato ◽  
2021 ◽  
Vol 2 (2) ◽  
pp. 167-181
Author(s):  
Marie Thérèse Rubio ◽  
Adèle Dhuyser ◽  
Stéphanie Nguyen
Keyword(s):  
Multiple Myeloma ◽  
Monoclonal Antibodies ◽  
Nk Cells ◽  
Tumor Cells ◽  
Nk Cell ◽  
Ex Vivo ◽  
Killer Cell ◽  
Proteasome Inhibitors ◽  
Disease Evolution ◽  
Cell Functions

Myeloma tumor cells are particularly dependent on their microenvironment and sensitive to cellular antitumor immune response, including natural killer (NK) cells. These later are essential innate lymphocytes implicated in the control of viral infections and cancers. Their cytotoxic activity is regulated by a balance between activating and inhibitory signals resulting from the complex interaction of surface receptors and their respective ligands. Myeloma disease evolution is associated with a progressive alteration of NK cell number, phenotype and cytotoxic functions. We review here the different therapeutic approaches that could restore or enhance NK cell functions in multiple myeloma. First, conventional treatments (immunomodulatory drugs-IMids and proteasome inhibitors) can enhance NK killing of tumor cells by modulating the expression of NK receptors and their corresponding ligands on NK and myeloma cells, respectively. Because of their ability to kill by antibody-dependent cell cytotoxicity, NK cells are important effectors involved in the efficacy of anti-myeloma monoclonal antibodies targeting the tumor antigens CD38, CS1 or BCMA. These complementary mechanisms support the more recent therapeutic combination of IMids or proteasome inhibitors to monoclonal antibodies. We finally discuss the ongoing development of new NK cell-based immunotherapies, such as ex vivo expanded killer cell immunoglobulin-like receptors (KIR)-mismatched NK cells, chimeric antigen receptors (CAR)-NK cells, check point and KIR inhibitors.

scholarly journals Cancer exosomes and natural killer cells dysfunction: biological roles, clinical significance and implications for immunotherapy

2022 ◽  
Vol 21 (1) ◽  
Author(s):  
Reza Hosseini ◽  
Hamzeh Sarvnaz ◽  
Maedeh Arabpour ◽  
Samira Molaei Ramshe ◽  
Leila Asef-Kabiri ◽  
...  
Keyword(s):  
Nk Cells ◽  
Natural Killer ◽  
Clinical Significance ◽  
Surveillance System ◽  
Cancer Biology ◽  
Nk Cell ◽  
Ex Vivo ◽  
Immune Surveillance ◽  
Tumor Development ◽  
Cell Responses

AbstractTumor-derived exosomes (TDEs) play pivotal roles in several aspects of cancer biology. It is now evident that TDEs also favor tumor growth by negatively affecting anti-tumor immunity. As important sentinels of immune surveillance system, natural killer (NK) cells can recognize malignant cells very early and counteract the tumor development and metastasis without a need for additional activation. Based on this rationale, adoptive transfer of ex vivo expanded NK cells/NK cell lines, such as NK-92 cells, has attracted great attention and is widely studied as a promising immunotherapy for cancer treatment. However, by exploiting various strategies, including secretion of exosomes, cancer cells are able to subvert NK cell responses. This paper reviews the roles of TDEs in cancer-induced NK cells impairments with mechanistic insights. The clinical significance and potential approaches to nullify the effects of TDEs on NK cells in cancer immunotherapy are also discussed.

scholarly journals Small Molecule Screening Identifies Rho-Associate Protein Kinase (ROCK) As a Regulator of NK Cell Cytotoxicity Against Cancer

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 3607-3607
Author(s):  
Grace Lee ◽  
Sheela Karunanithi ◽  
Zachary Jackson ◽  
David Wald
Keyword(s):  
Cell Therapy ◽  
Cytotoxic Activity ◽  
Nk Cells ◽  
Nk Cell ◽  
Ex Vivo ◽  
Akt Activation ◽  
Nk Cell Cytotoxicity ◽  
Rock Inhibition ◽  
Nk Cell Therapy

NK cells are a subset of lymphocytes that directly recognize and lyse tumor cells without the limitation of antigen specific receptor recognition. In addition to behaving as cytotoxic effector cells, NK cells unlike T cells are not thought to elicit graft versus host disease. The combination of these characteristics makes NK cells a powerful tool for adoptive cell therapy. Despite the promise of NK cell therapy, key hurdles in achieving significant clinical efficacy include both generating sufficient numbers of highly tumoricidal NK cells and maintaining the cytotoxic activity of these cells in vivo despite the immunosuppressive tumor microenvironment. Our lab and others have developed several feeder cell line-based expansion modules that robustly stimulate the ex vivo proliferation of NK cells. However, strategies to enhance and sustain the activity of NK cells once administered in vivo are still limited. In order to identify strategies to enhance the cytotoxic activity of NK cells, we developed a high-throughput small molecule screen (Figure 1A) that involved a calcein-based cytotoxicity assay of ex vivo expanded and treated NK cells against ovarian cancer cells (OVCAR-3). 20,000 compounds were screened and the screen was found to be highly robust (Z'&gt;0.59). We identified 29 hits that led to at least a 25% increase in cytotoxicity as compared to DMSO control-treated NK cells. One of the most promising hits was the pan-ROCK inhibitor, Y-27632 that led to an 30% increase in NK killing of the OVCAR-3 cells. We validated that ROCK inhibition leads to enhanced NK cell cytotoxic activity using Y-27632 (Figure 1B) as well as other well-established ROCK inhibitors such as Fasudil using a flow cytometry based killing assay. Y-27632 increased NK cell cytotoxicity in a dose- and time- dependent manner. ROCK inhibition consistently led to ~10-25% increase in NK cell cytotoxic activity directed against a variety of ovarian (Figure 1C) and other solid tumor cell lines (Figure 1D). Interestingly, we found that the NK hyperactivation persists for up to 48hrs after washing off the drug that may enable ex vivo stimulation before NK cell infusion. Our preliminary results showed that ROCK inhibition activates PI3K-dependent Akt activation (Figure 1E). We hypothesize that ROCK inhibition restores Akt activation which may be critical for NK cell activating receptor pathways and our current investigations will test these hypotheses. ROCK inhibitors, such as Y-27632 and Fasudil have been utilized in both preclinical and clinical studies for a variety of diseases such as atherosclerosis, neurodegenerative disorders, and ocular diseases. However, the consequences of ROCK inhibition in NK cells has not been thoroughly investigated. Our work shows a promising novel strategy to significantly enhance NK cell therapy against cancer that has high translational potential. Disclosures No relevant conflicts of interest to declare.

scholarly journals Combining Immunocytokine and Ex Vivo Activated NK Cells as a Platform for Enhancing Graft-Versus-Tumor Effects Against GD2+ Murine Neuroblastoma

2021 ◽  
Vol 12 ◽  
Author(s):  
Paul D. Bates ◽  
Alexander L. Rakhmilevich ◽  
Monica M. Cho ◽  
Myriam N. Bouchlaka ◽  
Seema L. Rao ◽  
...  
Keyword(s):  
Tumor Growth ◽  
Nk Cells ◽  
Nk Cell ◽  
Ex Vivo ◽  
Cell Transplant ◽  
Hematopoietic Stem ◽  
Allogeneic Hsct ◽  
Tnf Α

Management for high-risk neuroblastoma (NBL) has included autologous hematopoietic stem cell transplant (HSCT) and anti-GD2 immunotherapy, but survival remains around 50%. The aim of this study was to determine if allogeneic HSCT could serve as a platform for inducing a graft-versus-tumor (GVT) effect against NBL with combination immunocytokine and NK cells in a murine model. Lethally irradiated C57BL/6 (B6) x A/J recipients were transplanted with B6 bone marrow on Day +0. On day +10, allogeneic HSCT recipients were challenged with NXS2, a GD2+ NBL. On days +14-16, mice were treated with the anti-GD2 immunocytokine hu14.18-IL2. In select groups, hu14.18-IL2 was combined with infusions of B6 NK cells activated with IL-15/IL-15Rα and CD137L ex vivo. Allogeneic HSCT alone was insufficient to control NXS2 tumor growth, but the addition of hu14.18-IL2 controlled tumor growth and improved survival. Adoptive transfer of ex vivo CD137L/IL-15/IL-15Rα activated NK cells with or without hu14.18-IL2 exacerbated lethality. CD137L/IL-15/IL-15Rα activated NK cells showed enhanced cytotoxicity and produced high levels of TNF-α in vitro, but induced cytokine release syndrome (CRS) in vivo. Infusing Perforin-/- CD137L/IL-15/IL-15Rα activated NK cells had no impact on GVT, whereas TNF-α-/- CD137L/IL-15/IL-15Rα activated NK cells improved GVT by decreasing peripheral effector cell subsets while preserving tumor-infiltrating lymphocytes. Depletion of Ly49H+ NK cells also improved GVT. Using allogeneic HSCT for NBL is a viable platform for immunocytokines and ex vivo activated NK cell infusions, but must be balanced with induction of CRS. Regulation of TNFα or activating NK subsets may be needed to improve GVT effects.

Sign in / Sign up

Export Citation Format

Share Document