scholarly journals Insights into Clonal Relationships of Putative Adaptive Natural Killer Cells (NK) in Humans, Via Mapping of Somatic Piga Mutations in Patients with Paroxysmal Nocturna Hemoglobinuria (PNH)

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 2223-2223
Author(s):  
Thomas Winkler ◽  
Marcus A.F. Corat ◽  
Delong Liu ◽  
Moonjung Jung ◽  
Danielle M. Townsley ◽  
...  
Keyword(s):  
B Cells ◽  
Nk Cells ◽  
Peripheral Blood ◽  
Research Funding ◽  
Nk Cell ◽  
Myeloid Cells ◽  
Cell Subset ◽  
Loss Of Function ◽  
Hematopoietic Stem ◽  
Pnh Clone

Abstract NK cells play a central role in innate immunity, specifically in tumor surveillance and microbial pathogen control. Recent murine models and human studies have identified subsets of NK-cells with apparent memory cell function, strongly linked to CMV infection in humans and termed "adaptive" NK. Our recent clonal tracking studies following autologous hematopoietic stem cells transplantation (HSCT) of genetically-barcoded CD34+ cells in macaques revealed distinct clonal ontogeny of a subset of NK cells within the primate equivalent of the human CD56 dim population (Wu et al., Cell Stem Cell, 2014), with little clonal overlap with T-, B-lymphoid or myeloid cells, suggesting a separate precursor pool for this NK subtype. Peripheral blood CD 56bright -NK cells have been previously hypothesized to be precursors for the main population of circulating cytotoxic CD56dim cells. To further investigate NK-cell ontogeny and clonal relationships in humans we took advantage of naturally-occurring somatic mutations in the X-linked phosphatidylinositol glycan class A (PIGA) gene in patients with the hemolytic disorder paroxysmal nocturnal hemoglobinuria. This gene codes for an enzyme required for cell surface localization of glycosylphophatidylinositol (GPI)-anchored proteins, and thus loss of function mutations result in hematopoietic cells lacking GPI-anchored proteins, and red cell hemolysis. PNH patients have not been reported to have immune dysfunction and can have stable disease for many years. Membrane bound GPI anchors can be detected on any cell via flow cytometry using a labeled inactive aerolysin (FLAER), and serves as a marker for the fraction of cells comprising the PIGA mutant (GPI negative) clonal compartment. The PNH clone sizes contributing to peripheral blood cells are variable in but can reach almost 100% in some patients, and can be stable over decades. We selected 9 PNH patients with GPI negative granulocytes ranging from 5% to 98% and a median time from diagnosis of 43.7 months (15-100) for this study. NK cells were defined as CD56+/CD16+/CD3-/CD20- lymphocytes. We observed disproportionally fewer GPI negative NK cells compared to granulocytes (Fig 1), with the discrepancy most marked the major peripheral blood CD56 dim population (mean 65% vs 25% GPI negative granulocytes, p = 0.0028, paired t-test), in contrast to 46% GPI negative cells in the CD56bright population (p=0.057). Due to the prolonged life span of memory T and B cells, fewer GPI negative B and particularly T-lymphocytes have been reported in PNH patients. In our cohort 3.4% of CD3+ T-cells and 13.2% of CD20+ B-cells were GPI negative (p=0.0005 and 0.0014, respectively versus granulocytes). Compared to the NK subsets, the CD56 bright population showed the most significant differences (p = 0.0063 versus CD3 and p=0.0151 versus CD20). To further characterize the phenotype of the GPI positive versus negative CD56dim cells, we analyzed cells co-expressing either the terminal differential marker CD57, the inhibitory receptor NKG2A, or activating receptor NKG2C for FLAER positivity. Prior studies have suggested that the human CMV-linked adaptive NK subset is CD57+, NKG2A- and NKG2C+. The NKG2C+ CD 56dim population was highly enriched for GPI positive cells (p=0.0024 vs granulocytes, Fig 1). This profile was most prominent in CMV-IgG positive patients who had also significantly more GPI positive CD 56dim/CD57+ cells compared to granulocytes (p=0.008). Interestingly, one CMV positive patient (#5) had a complete lack of NKG2C expression, most likely due to homozygous loss of function mutation, and this patient had almost 100% GPI negative NK cells, matching his neutrophil pattern. Compared to granulocytes, NKGA2A+ or CD57+ positive CD56dim cells were also mostly GPI negative (p=0.0081 and 0.028). Circulating NK cell turnover has been estimated to be about 14 days. The PNH patients studied had documented clonal PIG-A mutations for many years. Our observation that the majority of CD56dim NK cells, specifically the NKG2C subset, are not progeny of the same progenitors producing CD56bright NK cells or myeloid cells based on clonal disparity regarding the PNH clone is suggestive of an independent, very long-lived or self-renewing NK cell progenitor for a CMV-linked CD56dim/CD57+/NKG2C+ memory NK cell compartment. These observations provide novel further insights into the human adaptive NK cell subset. Figure 1. Figure 1. Disclosures Winkler: Novartis: Research Funding; GSK: Research Funding. Townsley:Novartis: Research Funding; GSK: Research Funding.

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.

NK Cytotoxicity Is Decreased During the Early Posttransplant Period Following Pediatric Allogeneic Transplantation From Unrelated Donors Using CD3/CD19-Depleted Graft.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 2434-2434
Author(s):  
Antonio Pérez-Martínez ◽  
Manuel Ramírez ◽  
María Ruiz-Salmerón ◽  
Marta Gonzalez-Vicent ◽  
S. Grande ◽  
...  
Keyword(s):  
Stem Cell ◽  
Stem Cell Transplantation ◽  
Nk Cells ◽  
Cell Transplantation ◽  
Nk Cell ◽  
Cell Subset ◽  
Median Number ◽  
Hematopoietic Stem ◽  
Unrelated Donors ◽  
Nk Cytotoxicity

Abstract Abstract 2434 Poster Board II-411 Introduction and objectives: Unrelated donors, match unrelated (MUD) and haploidentical donors (HSCT), have been described as a therapeutic option for high-risk childhood acute leukemia. CD3/CD19 depleted graft has been used in order to decrease the incidence of graft versus host disease (GvHD) and post-transplant lymphoproliferative disease, in the unrelated transplantation setting. Donor-derived NK cell alloreactivity has been reported to mediate early graft-versus-leukemia (GvL) effect after allogeneic hematopoietic stem cell transplantation. NK cells are components of the innate immunity playing an important role in the surveillance of human tumors. NK cell recognition of malignant cells depends on a dynamic balance between activating and inhibitory receptors. NK cell alloreactivity can be predicted by donor Killer Immunoglobulin like Receptors (KIRs), Natural Killer Receptors (NCRs), C-type Lectin receptors (NKG2D), Toll Like Receptors (TLRs) and recipient human leukocyte antigen (HLA) class I alleles as ligands. Reduced risk of relapsed has been described in malignant cancer after haploidentical stem cell transplantation when HLA ligands against the inhibitory KIRs present in the donor were absent in the recipient (KIR–HLA receptor–ligand mismatch). We prospectively investigated NK function and NK reconstitution in 18 CD3/CD19 depleted graft unrelated hematopoietic stem cell transplantation (7 MUD and 11 HSCT) using fludarabine-based reduced intensity conditioning regimen. Results: NK cells peaked around day 30 after transplantation. The median number of NK cells on day +30 was 403±88/μL . On day 100 after transplantation the median number of NK cells/μL was 221±58. While the CD56bright NK cell subset was above normal during the first 100 days post-transplant, the “effector” NK cell subset, CD56dim CD16bright, was significantly reduced early after transplantation. The median percentage of CD56bright cells among NK cells in peripheral blood was 25.8±4.6% at day +30, and it was 24.5±5.7 at day +100. The decreased in CD56dim CD16bright NK cell subset was correlated with the decreased of the inhibitory KIR receptors (KIR2DL1, KIR2DL2, KIR3DL1) expression. We also observed a lower expression than donors of the activating receptors NKG2D, TLR4 at day +30, NKp46, TLR 9 at day 60 and NKp46, NKp30 at day +100. Although absolute NK-cell counts rapidly increased after transplant, their cytotoxicity against K562 was much lower compared to that of their donors. At day 100 after transplantation, patients NK cytotoxicity was lower than donor values. These results suggest that the low NK cell cytotoxicity could be related to an “immature” NK phenotype during the early period after HSCT. As other authors have published, activating receptors can be significantly upregulated in cytokine-stimulated NK cells. In our experience, overnight incubation with IL-15 overcomes this limitation, enhancing three times NK cytotoxicity, in vitro. Conclusion: The phenotype of NK cells and NK cytotoxicity ability are significantly altered early after allogeneic transplantation from unrelated donors using CD3/CD19-depleted graft. NK repertoire observed in patients was associated with the imbalance between CD56bright and CD56dim NK subsets and the expression of KIRs and NCRs. These data suggest a pattern consistent with an ongoing NK maturation after MUD and HSCT transplantation. In our experience, the phenotype and functional pattern of NK cells observed is suggestive of a cytokine-driven process. IL-15 stimulated NK cells could be helpful to optimize adoptive antitumor NK immunotherapy to enhance GvL effect as early as possible after transplantation. Disclosures: No relevant conflicts of interest to declare.

Natural Killer (NK) Cell Reconstitution After Haploidentical Unmanipulated Bone Marrow Transplantation with Reduced Intensity Conditioning

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 4557-4557 ◽  
Author(s):  
Isabel Gonzalez-Gascon y Marin ◽  
Ana Maria Perez-Corral ◽  
Jorge Gayoso ◽  
Javier Anguita ◽  
Cristina Pascual ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Stem Cell ◽  
Hematopoietic Stem Cell Transplantation ◽  
Nk Cells ◽  
Nk Cell ◽  
Cell Subset ◽  
Median Number ◽  
Reduced Intensity Conditioning ◽  
Hematopoietic Stem ◽  
Reduced Intensity

Abstract Abstract 4557 BACKGROUND: Natural killer (NK) cells are innate immune effectors that directly lyse virally infected or malignant cells. There are 2 different subsets of NK cells with distinct phenotypic and functional characteristics: the CD56dim subset, which composes 90% of peripheral blood NK cells and has a cytotoxic function, and the CD56bright subset, which cooperates with dendritic cells and T cells in lymph nodes to secrete interferon and promote adaptive immune responses. NK cells are the first donor-derived lymphocyte subset to reconstitute after hematopoietic stem cell transplantation, reaching normal levels after 1 month. Nearly all phenotyping studies of NK subsets after haploidentical hematopoietic stem cell transplantation (HHSCT) reveal a rapid reconstitution of NK cells towards the CD56bright subset. In addition, Y.-J. Chang et al found the highest 2-year survival in patients with a high number of CD56bright NK cells after unmanipulated HHSCT. We analyzed reconstitution of the NK compartment between days 90 and 180 after unmanipulated bone marrow HHSCT with reduced intensity conditioning (RIC). METHODS: Six adults received unmanipulated bone marrow HHSCT after RIC (fludarabine 30 mg/m2 [day –6 to –2], cyclophosphamide 14.5 mg/kg [day –6 and –5], and busulfan i.v. 3.2mg/kg [day –3]) at our institution between July 2007 and July 2010. Prophylaxis for acute graft-versus-host disease (GvHD) consisted of cyclophosphamide 50mg/kg (days +3 and +4) and cyclosporine A and mycophenolate mofetil from day +5 onwards. We monitored the reconstitution kinetics of circulating NK cells (CD56+, CD3–), and the CD56bright and CD56dim subsets by multiparametric flow cytometry (FC 500 Beckman® Coulter) at day +90 and day +180 after transplantation. Patient characteristics and clinical outcomes are shown in Table 1. 6 patients who underwent allogeneic HLA-identical sibling HSCT with RIC during the same period were used as controls. RESULTS: After HHSCT, NK cells reached normal levels in all patients but one at day +90, with a median number of NK cells of 111/mm3 (range, 25–195/mm3). At day +180 the median number of NK cells was 92/mm3 (range, 4–272/mm3). When we analyzed the absolute number of CD56bright and CD56dim subsets at day +90, we observed 2 patterns: Two patients showed skewed NK cell reconstitution towards CD56bright (Patient no. 3: 54 CD56bright/mm3; 11 CD56dim/mm3. Patient no. 4: 70 CD56bright/mm3; 17 CD56dim/mm3). Three patients reconstituted with a CD56dim/CD56bright ratio towards the CD56dim cell subset, similar to that of healthy adults (Patient no. 1: 17 CD56bright/mm3; 178 CD56dim/mm3. Patient no. 5: 9 CD56brigh/mm3; 135 CD56dim/mm3. Patient no. 6: 20 CD56bright/mm3; 116 CD56dim/mm3). One patient did not achieve adequate NK cell reconstitution (Patient no. 2: 15 CD56bright/mm3; 10 CD56dim/mm3). In contrast, in the control group, an increase in the CD56bright NK cell subset was not observed in any of the patients at any point. It is worth noting that 2 of the 3 patients with better clinical outcome (no GvHD, no relapse), namely patients no. 3 and no. 4 were the ones with skewed NK cell reconstitution towards the CD56bright NK cell subset. The other patient with a better clinical outcome (patient no. 6) had a normal CD56dim/CD56bright ratio at day +90. However, he showed an early CD56bright reconstitution (363 CD56bright/mm3; 34 CD56dim/mm3) in an additional determination on day +30. NK cell subsets reconstitution kinetics is shown in Figure 1. CONCLUSIONS: In our experience, NK cell reconstitution is adequate after RIC unmanipulated bone marrow HHSCT. Some patients recovered with a high proportion of CD56bright NK cells, as previously reported in other studies on HHSCT. Although limited by the sample size, our results are consistent with the previously observed survival advantage of patients with high early levels of CD56bright NK cells after unmanipulated haploidentical transplantation. Disclosures: No relevant conflicts of interest to declare.

CSL362: A Monoclonal Antibody to Human Interleukin-3 Receptor (CD123), Optimized for NK Cell-Mediated Cytotoxicity of AML Stem Cells

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 3598-3598 ◽  
Author(s):  
Samantha J. Busfield ◽  
Mark Biondo ◽  
Mae Wong ◽  
Hayley S. Ramshaw ◽  
Erwin M Lee ◽  
...  
Keyword(s):  
Stem Cells ◽  
Monoclonal Antibody ◽  
Mouse Model ◽  
Nk Cells ◽  
Peripheral Blood ◽  
Research Funding ◽  
Nk Cell ◽  
Target Cells ◽  
Cell Leukemia ◽  
Interleukin 3

Abstract Abstract 3598 The interleukin-3 receptor alpha chain (IL-3Rα/CD123) is expressed in a variety of hematological malignancies including AML, MDS, B-ALL, Hodgkin's lymphoma, hairy cell leukemia, systemic mastocytosis, plasmacytoid dendritic cell leukemia and CML. In AML, the majority of AML blasts express CD123 and this receptor is selectively over expressed on CD34+CD38− leukemic stem cells (LSC) compared to normal hematopoietic stem cells. This difference may provide a biological advantage to the leukemic cells given the survival and proliferation-promoting activities of IL-3, whilst at the same time providing an opportunity to target these malignant cells selectively. We have shown previously that 7G3, a mouse monoclonal antibody (mAb) which blocks IL-3 binding to CD123, is capable of eliminating human LSC in a mouse model of human AML by a combination of mechanisms, including engagement of the innate immune system via Fc-dependent mechanisms (Jin et al., 2009 Cell Stem Cell, 5:31). We have subsequently humanised and affinity-matured this antibody and, in addition, have engineered the Fc-domain to optimise potential cytotoxicity against AML cells. The resultant antibody, CSL362, retains the ability to neutralise IL-3 and has enhanced affinity for the FcγRIIIa (CD16) on NK cells. In vitro studies have demonstrated that the increased affinity for CD16 correlates with greater antibody-dependent cell-mediated cytotoxicity (ADCC) against CD123 expressing cell lines compared to CSL360, a non Fc-engineered anti-CD123 mAb. The improved activity was evident as both an increased maximal level of target cell lysis and as a shift in the EC50 of the antibody to lower concentrations. Importantly, both primary AML blasts and CD34+CD38−CD123+LSC were susceptible to CSL362-induced ADCC and this was seen even in samples that were resistant to ADCC by a non Fc-engineered anti-CD123 mAb. In an AML xenograft mouse model, where treatment with the antibody was initiated 4 weeks after engraftment of leukemia cells, CSL362 was more effective in reducing leukemic growth than the non Fc-engineered anti-CD123 mAb. The evaluation of neutrophils, monocytes, macrophages and NK cells in ADCC assays has revealed that the major effector cell responsible for CSL362-mediated cytotoxicity in human peripheral blood is the NK cell. In clinical samples we have been able to demonstrate autologous depletion ex vivo of target AML blasts (collected at diagnosis and cryopreserved) following incubation with CSL362 and peripheral blood mononuclear cells (taken from the same patient at first remission), indicating that NK cell number and function is sufficiently preserved in such patients for CSL362-directed killing of leukemic target cells. The pre-clinical data generated thus far strongly support the clinical development of CSL362 for the treatment of AML in patients with adequate NK cell function. A Phase 1 study of CSL362 in patients with CD123 positive AML in remission is underway (Clinical Trials.gov identifier: NCT01632852). Disclosures: Busfield: CSL Limited: Employment. Biondo:CSL Limited: Employment. Wong:CSL Limited: Employment. Ramshaw:CSL Limited: Research Funding. Lee:CSL Limited: Research Funding. Martin:CSL Limited: Employment. Ghosh:CSL Limited: Employment. Braley:CSL Limited: Employment. Tomasetig:CSL Limited: Employment. Panousis:CSL Limited: Employment. Vairo:CSL Limited: Employment. Roberts:CSL Limited: Research Funding. DeWitte:CSL Behring: Employment. Lock:CSL Limited: Consultancy, Research Funding. Lopez:CSL Limited: Consultancy, Research Funding. Nash:CSL Limited: Employment.

The Influence Of KIR Haplotype In ITP Incidence, Treatment Response and Bleeding Symptoms

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 2316-2316
Author(s):  
Bethan Psaila ◽  
Nayla Boulad ◽  
Emily Leven ◽  
Naznin Haq ◽  
Christina Soo Lee ◽  
...  
Keyword(s):  
Platelet Count ◽  
B Cells ◽  
Nk Cells ◽  
Board Of Directors ◽  
Research Funding ◽  
Nk Cell ◽  
Advisory Committees ◽  
Kir Genes ◽  
Number Of Patients

Abstract The pathogenesis of immune thrombocytopenia (ITP) is multifactorial, with both cellular and humoural immune dysfunction. The role of NK cells has not been well defined in ITP but in other diseases NK cells have a role in rejecting “foreign” eg transplanted organ or tumor, and also acting against self as occurs in autoimmunity. NK cell activity is orchestrated by the balance of activating vs. inhibitory signalling, in particular via the killer cell immunoglobulin-like receptor (KIR) family of receptors. Significant variation exists in KIR allelic subtype and copy number for the KIR between individuals, and associations have been made with certain haplotypes and a number of autoimmune disorders including rheumatoid arthritis, scleroderma and diabetes. Previous reports have demonstrated a reduction in natural killer (NK) cell number and function in ITP and expression of inhibitory KIR genes is increased in patients in remission vs. active ITP. Methods To explore whether a particular KIR haplotype might predispose to ITP, and also affect response to ITP treatment, we performed KIR genotyping using the Invitrogen SSP kit on 92 patients attending a haematology centre in New York and compared the results to data from 213 controls taken from the USA Eastern Database. Genomic DNA was typed for the inhibitory KIR genes KIR2DL1, KIR2DL2, KIR2DL5A (alleles 001 and 002), KIR2DL5B (alleles 002-004, 06, and 007), KIR3DL1, KIR3DL3; the activating KIR genes KIR2DS1, KIR2DS2, KIR2DS3, KIR2DS4, KIR2DS5, KIR3DS1; the framework genes KIR2DL3, KIR2DL4, KIR3DL2, KIR3DP1; and the pseudogene KIR2DP1. The patients with ITP had been or were receiving treatment with IVIG (n=64), corticosteroids (72) and rituximab (37). Bleeding symptoms were recorded. Response to treatment was defined as complete - platelet count increase to > 100 x 109/mL; partial - platelet count increase to > 50 x 109/mL; or no response. For the purpose of analysis, PRs and CRs were combined. A comprehensive database allowed a logistic regression, assessing both responses to treatments, platelet counts, neutrophil counts, CRP, lymphocyte subsets and bleeding symptoms. Results The expression of two inhibitory KIR genes, 2DL1 and 3DL1, was significantly lower in the patients with ITP as compared to controls (87% 2DL1 and 87% 3DL1 compared to 99% in controls - P < 0.02). Response to rituximab was strongly related to KIR haplotype expression. 2DL1 expression was higher among nonresponders to Rituximab (100% of non responders compared to 82% of responders), whereas 2DL3 expression was significantly lower (79% compared to 90%) (P < 0.05, Figure 1B). Separately, patients with the 2DS3 allele, an activatory KIR, were 5.5 times more likely to have experienced significant bleeding. Conclusions Although these findings are preliminary and require further investigation, these data suggest that increased cytotoxic autoimmunity due to reduced KIR inhibition may be associated with the development of ITP and possibly contribute importantly to the pathogenesis. Anti-CD20 targeting therapy directed at B cells was strongly influenced by 2 different KIRs (1 upregulated and one down-regulated) emphasizing the potential role of NK cells in elimination of tissue-based (nodal) B cells. Finally a more pronounced clinical phenotype with a markedly higher incidence of severe bleeding associated with an increased activatory KIR expression demonstrates the role of NK cells in bleeding presumably via their effects on either endothelial cells or platelet function. These exciting findings will be pursued for confirmation in a larger number of patients. Disclosures: Bussel: Amgen: Family owns stock Other, Membership on an entity’s Board of Directors or advisory committees, Research Funding; Cangene: Research Funding; Genzyme: Research Funding; GlaxoSmithKline: Family owns stock, Family owns stock Other, Membership on an entity’s Board of Directors or advisory committees, Research Funding; IgG of America: Research Funding; Immunomedics: Research Funding; Ligand: Membership on an entity’s Board of Directors or advisory committees, Research Funding; Eisai: Membership on an entity’s Board of Directors or advisory committees, Research Funding; Shionogi: Membership on an entity’s Board of Directors or advisory committees, Research Funding; Sysmex: Research Funding; Symphogen: Membership on an entity’s Board of Directors or advisory committees.

Chronic Myeloid Leukemia Patients with Deep Molecular Responses to Tyrosine Kinase Inhibitors Have Increased Effector Natural Killer and Cytotoxic T Cell Immune Responses to Leukaemia-Associated Antigens and Concomitant Reduced Immune Suppressors

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 18-18 ◽  
Author(s):  
Amy Hughes ◽  
Carine Tang ◽  
Jade Clarson ◽  
Ljiljana Vidovic ◽  
Timothy P. Hughes ◽  
...  
Keyword(s):  
T Cells ◽  
B Cells ◽  
Immune Responses ◽  
Nk Cells ◽  
Research Funding ◽  
Kinase Inhibitors ◽  
Nk Cell ◽  
Molecular Response ◽  
Molecular Responses ◽  
B Cell Subsets

Abstract We hypothesized that immune responses contribute to deep BCR-ABL molecular responses in chronic phase chronic myeloid leukaemia (CML) patients on tyrosine kinase inhibitors (TKI). We studied 32 CML patients; 16 at diagnosis, patients treated with imatinib (n=20), nilotinib (n=9) or dasatinib (n=3). Methodology: The effector immune responses of Natural Killer (NK) cells were characterized by flowcytometry and functional analysis by CD107a degranulation assay. Cytotoxic T lymphocyte (CTL) responses to leukaemia-associated antigens (LAAs) WT1, BMI-1, PR3 and PRAME were quantified by interferon-gamma ELISPOT using peptide libraries of 15-mer peptides overlapping by 11 amino acids spanning the entire protein, or HLA-A0201 specific peptides in HLA-A0201+ patients. Immune suppressor regulatory T cells (Treg), Myeloid Derived Suppressor Cells (MDSC), Programmed cell death-1 (PD-1) expression on T cells, NK cells, B cells and monocytes, and major B cell subsets were extensively characterized by flowcytometry. Results: Patients in deep molecular response (MR4.5; BCR-ABL <0.0032%) displayed increased antigen-specific CTL responses to LAAs, both in the number of positive LAAs and frequency of responses, compared to patients at diagnosis and major molecular response (MMR; BCR-ABL <0.1%). The most abundant LAA response was to PRAME [51% of patients in MR4.5 compared to 31% in MMR and 0% at diagnosis] and WT1 [31% of patients in MR4.5 compared to 28% in MMR and 0% at diagnosis]. PR3-specific immune responses were the least abundant, with no difference in response between MR4.5and MMR (both 3%) compared to 0% at diagnosis. Immunophenotypic analysis revealed a shift toward a more mature, cytolytic NK cell phenotype (CD57+, CD161+CD62L-) in MMR and MR4.5, consistent with up-regulation of the CD94/NKG2 family of inhibitory/activating receptors (NKG2A, NKG2C and NKG2D), the cytotoxicity triggering receptor NKp46 and a functional increase in NK cell cytotoxicity capacity against K562 target cells. The percentage of CD3-CD56dimCD16bright cytolytic NK cells as a proportion of total lymphocytes was significantly increased in MMR and MR4.5 [33.6% ± 6.6 p=0.0008 and 33.1% ± 4.1 p=0.01, respectively] compared to 7.8% ± 2.8 at diagnosis. The absolute Treg number/µl was significantly lower in patients in MMR and MR4.5 [13.9 ± 1.7 and 10 ± 1.1, respectively] compared to 32.7 ± 4.4 at diagnosis. Similarly, MDSC were significantly reduced in patients in MMR and MR4.5 [3.9 ± 0.9 and 1.9 ± 0.5 MDSC/µl] compared to diagnosis [18.3 ± 3.9]. A predominantly granulocytic (CD66b+CD15+) MDSC phenotype was seen in CML patients at diagnosis. PD-1 expression as a proportion of total lymphocytes was significantly decreased in cytotoxic CD8+ T cells in MR4.5 [5.7% ± 1.2] compared to MMR [12.3% ± 2.0, p=0.008] and patients at diagnosis [21.7% ± 5.2, p=0.0003]. PD-1 expression was decreased in CD4+ helper T cells in MR4.5 [7.5% ± 1.7] compared to MMR [11.4% ± 1.5, p=0.07] and diagnosis [17% ± 2.9, p=0.008]. Overall, PD-1 expression was lower in NK cells in CML patients, albeit significant in MMR and MR4.5 [0.24% ± 0.09, p=0.006 and 0.36 ± 0.07, p=0.02, respectively] compared to [1.42% ± 0.4] at diagnosis. No difference in PD-1 expression was seen in B cells or monocytes. No significant difference was observed in CD3-CD19+ B cells in MMR and MR4.5 or at diagnosis. Analysis of major B cell subsets revealed no difference in the proportion of transitional, naïve or memory B cells, plasma blasts or plasma cells. Conclusion: Enhanced effector immune responses of NK and LAA-specific CTLs are associated with concomitant reduction in immune suppressor activity, and may increase the rate of deep molecular responses to TKIs in CML. Methods to augment these responses may result in greater rate of success in TKI cessation studies. Disclosures Hughes: ARIAD: Honoraria, Research Funding; Bristol-Myers Squibb: Honoraria, Research Funding; Novartis: Honoraria, Research Funding. Yong:Novartis: Honoraria, Research Funding.

scholarly journals Engineered Interleukin-15 Autocrine Signaling Invigorates Anti-CD123 CAR-NK Cells

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 2806-2806
Author(s):  
Ilias Christodoulou ◽  
Michael Koldobskiy ◽  
Won Jin Ho ◽  
Andrew Marple ◽  
Wesley J. Ravich ◽  
...  
Keyword(s):  
Nk Cells ◽  
Peripheral Blood ◽  
Research Funding ◽  
Nk Cell ◽  
Xenograft Model ◽  
Blood Analysis ◽  
Target Cells ◽  
Interleukin 15 ◽  
Peripheral Blood Analysis

Abstract Introduction : Acute Myeloid Leukemia (AML) is an aggressive neoplastic disorder with poor outcomes in children and adults. NK cell adoptive transfer is an anti-cancer immunotherapy that has promise for AML treatment. We aimed to improve NK cell anti-tumor efficacy with expression of a Chimeric Antigen Receptor (CAR) on the cell surface. Our CAR consists of an extracellular single-chain variable fragment targeting the AML-associated antigen CD123 (IL3Rα) and intracellular domains derived from 2B4 and TCRζ. We sought to improve the persistence and long-term functionality of our CAR-NKs by introducing transgenic interleukin-15 (IL15). Methods: CD3-depleted PBMCs were first activated with lethally irradiated feeder cells, then transduced with transiently produced replication incompetent γ-retrovirus (αCD123.2B4.ζ, αCD123.2B4.ζ-IRES-sIL15, sIL15-IRES-mOrange) on day 4 of culture. CAR expression was measured on day 8 using FACS. Secretion of IL15 was verified with ELISA. Cytotoxicity was measured using ffLuc expressing target cells and bioluminescence (BL) measurement. In serial stimulation assays, target cells were repleted daily to maintain a 1:1 effector:target ratio. Immunophenotype and cell counts were assessed by FACS. Transcriptomic analysis (RNAseq) was performed on RNA derived from NK cells purified on D10. Xenograft modeling was performed using NSG mice engrafted with MV-4-11.ffLuc or MOLM-13.ffLuc AML cell lines. Mice were treated with NK cells on D4 or D4-7-10. Untreated mice served as controls. Tumor growth was serially tracked in vivo using BL imaging. NK cell persistence and expansion were measured in peripheral blood. Results: The 2B4.ζ CAR was well expressed on the surface of transduced NK cells (median transduction efficiency 95%, range 85-97%, n=3). 2B4.ζ CAR-NK treatment prolonged survival of AML engrafted mice when compared to treatment with unmodified NKs (median survival: 63 vs 55 days; n=8 mice; p=0.014). Serial peripheral blood analysis revealed a steady decline in circulating NK cells, which were undetectable in all cohorts within 21 days. NK cells were then engineered for constitutive secretion of IL15, with and without CAR expression. 2B4.ζ/sIL15 CAR-NKs had the most potent 24h-cytotoxicity against CD123+ targets (Fig. 1). After a 10-day chronic stimulation with MV-4-11, 2B4.ζ/sIL15- and sIL15-NKs expanded (x1.2 and x5.9 respectively), while NK cells without sIL15 decreased in number. In this assay, only 2B4.ζ/sIL15 CAR-NKs exhibited sustained tumor killing. Transcriptomic analysis after 10 days of serial stimulation showed sample clustering dependent on IL15 secretion. Differential gene expression analysis (DESeq2) identified upregulation of genes associated with cell cycle progression, apoptosis regulation, chemokine signaling, and NK cell mediated cytotoxicity in NK cells secreting IL15 compared to those without. In multiparameter flow cytometric analysis, 2B4.ζ/sIL15 CAR-NKs had a higher percentage of NK cells populating clusters defined by higher surface expression of NK cell activating receptors (NKp30, NKG2D, LFA-1) compared to 2B4.ζ and unmodified NK cells. In our MV-4-11 xenograft model, NKs armed with secreted IL15 expanded in vivo and had improved persistence. A single dose (D4) of 2B4.ζ/sIL15 CAR-NKs demonstrated an initial antitumor response, equivalent to that seen following 3 doses (D4-7-10) of 2B4.ζ CAR-NKs. However, mice treated with IL15-secreting NKs had short survival (Fig. 2). Compared to control mice, peripheral blood analysis showed increasing systemic hIL15 and higher levels of hTNFα. In our more aggressive MOLM-13 xenograft model, both single dose 2B4.ζ/sIL15 CAR-NK and multiple dose 2B4.ζ CAR-NK treatment prolonged survival compared to treatment with unmodified NKs. (27 and 26 vs 20 days; n=5 mice; p&lt;0.01; Fig. 2). Conclusion: 2B4.ζ CAR-NKs have limited antitumor efficacy and short persistence in vivo. NK cells armored with secreted IL15 have enhanced anti-AML cytotoxicity and in vitro persistence. Introduction of IL15 secretion confers a distinctly activated phenotype that is maintained during chronic antigen stimulation. Constitutive local IL15 secretion improves in vivo NK cell persistence but may cause lethal toxicity when employed against AML. These results warrant further study and should impact the development of CAR-NK clinical products for patients with AML. Figure 1 Figure 1. Disclosures Ho: Rodeo Therapeutics/Amgen: Patents & Royalties; Exelixis: Consultancy; Sanofi: Research Funding. Bonifant: Kiadis Pharma: Research Funding; BMS: Research Funding; Merck, Sharpe, Dohme: Research Funding.

scholarly journals Off-the-Shelf, Multiplexed-Engineered iPSC-Derived NK Cells Mediate Potent Multi-Antigen Targeting of B-Cell Malignancies with Reduced Cytotoxicity Against Healthy B Cells

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 407-407
Author(s):  
Frank Cichocki ◽  
Jode P Goodridge ◽  
Ryan Bjordahl ◽  
Svetlana Gaidarova ◽  
Sajid Mahmood ◽  
...  
Keyword(s):  
T Cells ◽  
B Cells ◽  
Nk Cells ◽  
Board Of Directors ◽  
Research Funding ◽  
Nk Cell ◽  
Advisory Committees ◽  
Car T Cells ◽  
Car T

Abstract Treatments for B-cell malignancies have improved over the past several decades with clinical application of the CD20-specific antibody rituximab and chimeric antigen receptor (CAR) T cells targeting CD19. Despite the success of these therapies, loss of CD20 after rituximab treatment has been reported in leukemia and lymphoma patients. Additionally, up to 50% of all patients receiving anti-CD19 CAR T-cell therapy relapse within the first year with many of those patients exhibiting CD19 loss. Thus, new therapeutic approaches are needed to address tumor antigen escape. Accordingly, we generated triple gene-modified iPSC-derived NK (iNK) cells, termed "iDuo" NK cells, tailored to facilitate multi-antigen targeting. The iPSC line was clonally engineered to express high-affinity, non-cleavable CD16a (hnCD16), an anti-CD19 CAR optimized for NK cell signaling, and a membrane-bound IL-15/IL-15R fusion (IL-15RF) molecule to enhance NK cell persistence (Fig. 1A). To model antigen escape, we generated CD19 knockout AHR77 lymphoma cells alongside wild type AHR77 cells (both CD20 +) as targets in cytotoxicity assays. Activated peripheral blood NK (PBNK) cells, non-transduced iNK cells, and iDuo NK cells were tested as effectors. Unlike PBNK cells or non-transduced iNK cells, iDuo NK cells efficiently eliminated wild type AHR77 cells with or without the addition of rituximab at all tested E:T ratios. Similarly, iDuo NK cells in combination with rituximab were uniquely able to efficiently eliminate CD19 KO AHR77 cells due to enhanced antibody-dependent cellular cytotoxicity (ADCC) driven by hnCD16 (Fig. 1B-E). Cytotoxicity mediated by iDuo NK cells was also evaluated using primary chronic lymphocytic leukemia (CLL) cells. Compared to expanded PBNK cells and non-transduced iNK cells, only iDuo NK cells (in the absence of rituximab) were able to kill primary CLL cells (Fig. 1F). Expression of IL-15RF by iDuo NK cells uniquely supports in vitro expansion without the need for cytokine supplementation. To determine whether IL-15RF supports in vivo persistence of iDuo NK cells, CD19 CAR iNK cells (lacking IL-15RF) and iDuo NK cells were injected into NSG mice without the addition of cytokines or CD19 antigen availability. iDuo NK cell numbers peaked within a week after injection and persisted at measurable levels for ~5 weeks, in marked contrast to CD19 CAR iNK cell numbers that were undetectable throughout (Fig. 1G). To evaluate the in vivo function of iDuo NK cells, NALM6 leukemia cells were engrafted into NSG mice. Groups of mice received tumor alone or were treated with 3 doses of thawed iDuo NK cells. iDuo NK cells alone were highly effective in this model as evidenced by complete survival of mice in the treatment group (Fig. 1H). To assess iDuo NK cells in a more aggressive model, Raji lymphoma cells were engrafted, and groups of mice received rituximab alone, iDuo NK cells alone, or iDuo NK cells plus rituximab. Mice given the combination of iDuo NK cells and rituximab provided extended survival compared to all other arms in the aggressive disseminated Raji lymphoma xenograft model (Fig. 1I). One disadvantage of anti-CD19 CAR T cells is their inability to discriminate between healthy and malignant B cells. Because NK cells express inhibitory receptors that enable "self" versus "non-self" discrimination, we reasoned that iDuo NK cells could have higher cytotoxicity against tumor cells relative to healthy B cells. To address this, we labeled Raji cells, CD19 + B cells from healthy donor peripheral blood mononuclear cells (PBMCs) and CD19 - PBMCs. Labeled populations of cells were co-cultured with iDuo NK cells, and specific killing was analyzed. As expected, iDuo NK cells did not target CD19 - PBMCs. Intriguingly, iDuo NK cells had much higher cytotoxic activity against Raji cells compared to primary CD19 + B cells, suggesting a preferential targeting of malignant B cells compared to healthy B cells. Together, these results demonstrate the potent multi-antigen targeting capability and in vivo antitumor function of iDuo NK cells. Further, these data suggest that iDuo NK cells may have an additional advantage over anti-CD19 CAR T cells by discriminating between healthy and malignant B cells. The first iDuo NK cell, FT596, is currently being tested in a Phase I clinical trial (NCT04245722) for the treatment of B-cell lymphoma. Figure 1 Figure 1. Disclosures Cichocki: Gamida Cell: Research Funding; Fate Therapeutics, Inc: Patents & Royalties, Research Funding. Bjordahl: Fate Therapeutics: Current Employment. Gaidarova: Fate Therapeutics, Inc: Current Employment. Abujarour: Fate Therapeutics, Inc.: Current Employment. Rogers: Fate Therapeutics, Inc: Current Employment. Huffman: Fate Therapeutics, Inc: Current Employment. Lee: Fate Therapeutics, Inc: Current Employment. Szabo: Fate Therapeutics, Inc: Current Employment. Wong: BMS: Current equity holder in publicly-traded company; Fate Therapeutics, Inc: Current Employment. Cooley: Fate Therapeutics, Inc: Current Employment. Valamehr: Fate Therapeutics, Inc.: Current Employment. Miller: Magenta: Membership on an entity's Board of Directors or advisory committees; ONK Therapeutics: Honoraria, Membership on an entity's Board of Directors or advisory committees; Vycellix: Consultancy; GT Biopharma: Consultancy, Patents & Royalties, Research Funding; Fate Therapeutics, Inc: Consultancy, Patents & Royalties, Research Funding; Sanofi: Membership on an entity's Board of Directors or advisory committees; Wugen: Membership on an entity's Board of Directors or advisory committees.

scholarly journals CD16-IL15-CLEC12A Trispecific Killer Engager (TriKE) Drives NK Cell Expansion, Activation, and Antigen Specific Killing of Cancer Stem Cells in Acute Myeloid Leukemia

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 1454-1454 ◽  
Author(s):  
Upasana Sunil Arvindam ◽  
Paulien van Hauten ◽  
Caroline Hallstrom ◽  
Daniel A. Vallera ◽  
Harry Dolstra ◽  
...  
Keyword(s):  
Stem Cells ◽  
Nk Cells ◽  
Myeloid Leukemia ◽  
Research Funding ◽  
Nk Cell ◽  
Target Cells ◽  
Hematopoietic Stem ◽  
Functional Assays ◽  
Target Effects

Abstract Our group developed a 161533 trispecific killer engager (TriKE) molecule to target acute myeloid leukemia (AML) cells using Natural Killer (NK) cells. This molecule contains an anti-CD16 camelid nanobody to activate NK cells, an anti-CD33 single chain variable fragment (scFv) to engage cancer targets, and an IL-15 molecule that drives NK cell priming, expansion and survival. Using an earlier version of this molecule, we have shown that the CD33 TriKE is effective at activating NK cells against AML targets in vitro and in vivo. This preclinical data has lead to the establishment of a clinical trial in refractory AML patients at the University of Minnesota, set to open Q3 2018. While these previous studies have validated the use of TriKEs as an effective strategy of harnessing NK cells in cancer immunotherapy, CD33 has limitations as a target antigen. The high mortality and poor five-year survival rates (26%) for AML patients can be attributed to chemotherapy resistance and disease relapse. A majority of chemotherapy resistant leukemia stem cells (LSCs), that are hypothesized to facilitate relapse, do not express CD33. In addition, all hematopoietic stem cells and normal myeloid cells express CD33, thus targeting this antigen can lead to severe defects in hematopoiesis and on-target/off-tumor toxicity. To address these limitations, we developed a TriKE that targets CLEC12A or C-type lectin-like molecule 1 (CLL-1). CLEC12A is highly expressed on AML cells and over 70% of CD33 negative cells express CLEC12A. It has been attributed as a stem cell marker in AML, being selectively overexpressed in LSCs. CLEC12A is expressed by CD34+/CD38- LSCs but not normal CD34+/CD38- hematopoietic stem cells in regenerating bone marrow, thus minimizing off-target effects. The CLEC12A TriKE was developed in a mammalian cell system to ensure that appropriate post-translational modifications are present. We confirmed that the TriKE binds specifically to HL-60 and THP-1 target cells that express CLEC12A compared to Raji cells that do not express CLEC12A. Treatment of peripheral blood mononuclear cells (PBMCs) with the CLEC12A TriKE drives a significant increase in NK cell specific proliferation over 7 days as measured by CellTrace dilution compared to treatment with a CLEC12A scFv or IL-15 alone (69.7 ± 6.7% vs 11.9 ± 2.5% vs 38.4 ± 7.3%) (Figure 1A). To measure NK cell killing, we conducted an IncuCyte Zoom assay. Here, HL-60 target cells were labeled with a caspase 3/7 reagent where a color change indicates target cell death. The CLEC12A TriKE was able to induce more target cell killing than CLEC12A scFv or IL-15 as measured by number of live target cells at the end of the 48 hour assay (53.9 ± 1.9% vs 103.3 ± 3.4% vs 71.1 ± 1.4%). The CLEC12A TriKE induces an increase in NK cell degranulation, measured by CD107a expression against HL-60 AML tumor targets in a 4 hour functional assay compared to treatment with CLEC12A scFv or IL-15 alone (62.3 ± 1.1% vs 19.4 ± 3.8% vs 27.5 ± 4.9%). In this assay, there is also an increase in cytokine production, measured by IFNg and TNFa respectively (16.7 ± 4.2% vs 2.3 ± 1.5% vs 4.7 ± 1.9% and 18.0 ± 5.1% vs 2.5 ± 1.7% vs 4.6 ± 2.5%) (Figure 1B). We observe a similar enhanced functional response with THP-1 AML tumor targets. In these functional assays, treatment with the CLEC12A TriKE produced less background activation compared to the CD33 TriKE, indicating less off-target effects on PBMCs. To confirm the clinical relevance of this molecule, we tested the efficacy of the CLEC12A TriKE against primary AML targets. AML blasts were identified as SSC low, CD45 intermediate and CD34 high cells. Out of the 9 AML samples tested, 7 expressed high levels of CD33 (70.4 ± 6.3%) and CLEC12A (78.1 ± 5.2%). In functional assays with these samples, the CLEC12A TriKE was able to induce greater CD107a and IFNg expression, and enhanced killing of tumor targets as measured by a live/dead stain compared to CLEC12A scFv or IL-15 (Figure 1C). In these assays, the efficacy of the CLEC12A TriKE was comparable to the CD33 TriKE. Our data demonstrates that the CLEC12A TriKE drives NK cell specific proliferation, degranulation, cytokine secretion, and killing of tumor targets in vitro. Apart from AML, CLEC12A is expressed on cancer cells and LSCs in patients with myelodysplastic syndromes (MDS). These findings highlight the clinical potential of the CLEC12A TriKE individually or in combination with the CD33 TriKE for the treatment of MDS and AML. Figure 1. Figure 1. Disclosures Vallera: GT Biopharma: Consultancy, Research Funding. Felices:GT Biopharma: Research Funding.

scholarly journals Selinexor Enhances NK Cell Activation Against Lymphoma Cells Via Downregulation of HLA-E

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 2411-2411
Author(s):  
Jack Fisher ◽  
Christopher J. Walker ◽  
Peter Johnson ◽  
Mark S Cragg ◽  
Francesco Forconi ◽  
...  
Keyword(s):  
Nk Cells ◽  
Peripheral Blood ◽  
Research Funding ◽  
Cell Activation ◽  
Nk Cell ◽  
Cell Activity ◽  
Fold Increase ◽  
Target Cells ◽  
Lymphoma Cells ◽  
Pre Treatment

Abstract Introduction: Natural killer (NK) cells are powerful immune effectors which induce direct cytotoxicity, promote adaptive immune responses and mediate antibody dependent cellular cytotoxicity (ADCC). Enhancement of NK cell activity against cancer is currently the focus of intense research efforts and strategies include CAR-NK, stimulatory antibodies, cytokines and checkpoint inhibitors. Upregulation of exportin-1 (XPO1) is common in human cancers and high expression is negatively associated with survival in various cancers including diffuse large B cell lymphoma (DLBCL). Targeted inhibition of XPO1 by the selective inhibitor selinexor leads to cancer cell death via accumulation of tumour suppressor proteins in the nucleus, dysregulation of growth regulatory proteins and blockade of oncogene protein translation. The therapeutic efficacy of XPO1 inhibition has led to FDA approval of the oral XPO1 inhibitor selinexor for the treatment of multiple myeloma and DLBCL. The effect of selinexor on NK cell activity has not previously been investigated and was therefore addressed in this study. Methods: The B lymphoma cell lines JeKo-1, SU-DHL-4 and Ramos were incubated with selinexor (50-2000nM) for 18 hours before analysis. Flow cytometry was used to assess cell surface expression of activating and inhibitory ligands for NK cells. For NK based assays, peripheral blood derived NK cells were isolated from healthy donors and incubated with IL-15 (1ng/ml) overnight prior to co-culture with target lymphoma cells for a further 4 hours. Cytotoxicity was assessed using propidium iodide staining of target cells and degranulation of NK cells was assessed by measurement of CD107a. Whole blood samples from colorectal cancer patients (n=11) at pre-treatment and 3 weeks post selinexor monotherapy were assessed by flow cytometry for CD45+CD3-CD19-CD56+ NK cells. Results: Selinexor pre-treatment of target lymphoma cells significantly increased NK cell mediated cytotoxicity against SU-DHL-4 (2.2 Fold increase, p&lt;0.01), JeKo-1 (2 Fold increase, p&lt;0.01) and Ramos (1.7 Fold increase, p&lt;0.01) cells. In accordance with this, selinexor pre-treatment of target cells also increased the activation of NK cells against SU-DHL-4, JeKo-1 and Ramos cells as measured by CD107a expression in both CD56 bright and CD56 dim NK cell sub-groups. To identify the mechanism behind this, we measured expression of activating and inhibitory ligands for NK cells on SU-DHL-4 cells after incubation with selinexor. No significant changes in expression of activating ligands (MICA/B, ULBP-2/5/6, ULBP-1, Vimentin, B7H6, CD54) were evident. In contrast, selinexor significantly (p&lt;0.001) reduced the surface expression of HLA-E on SU-DHL-4 cells by 50%. Selinexor mediated downregulation of HLA-E was also evident in Ramos (60% reduction, p&lt;0.001) and JeKo-1 cells (20% reduction, p&lt;0.01). HLA-E binds the ITIM containing receptor NKG2A, a key inhibitory receptor for NK cells and subsets of T cells. In accordance with this, selinexor pre-treatment of SU-DHL-4 cells selectively increased NKG2A+ NK cell activation (p&lt;0.01) following co-culture. To examine the effect of selinexor on NK cells in patients, we assessed the proportion of NK cells in the peripheral blood of 11 colorectal cancer patients at pre-treatment and three weeks post selinexor monotherapy. % NK cells of CD45+ peripheral blood lymphocytes following treatment with selinexor was increased 2-fold (Median 5% pre-treatment vs 10% post selinexor). In addition, increased abundance of the less mature and less cytotoxic CD56 bright subset of NK cells was associated with poor response to therapy (Median 4% responders (n=3) vs 20% non-responders (n=8)). Larger patient datasets are required to confirm these effects and this analysis is currently ongoing. The effect of selinexor on NK cells in patients with lymphoma is also currently under investigation. Conclusions: The NKG2A:HLA-E axis is a novel immune checkpoint target and our data identifies that selinexor sensitises lymphoma cells to NK cell mediated killing via disruption of this interaction. In addition, we provide initial evidence that NK cells may be associated with clinical response to selinexor. This data indicates that NK cells may contribute to the therapeutic efficacy of selinexor and that selinexor may synergise with NK cell targeted therapies for the treatment of lymphoma. Disclosures Walker: Karyopharm Therapeutics: Current Employment, Current holder of individual stocks in a privately-held company, Current holder of stock options in a privately-held company. Johnson: Morphosys: Honoraria; Kymera: Honoraria; Kite Pharma: Honoraria; Incyte: Honoraria; Genmab: Honoraria; Celgene: Honoraria; Bristol-Myers: Honoraria; Epizyme: Consultancy, Research Funding; Boehringer Ingelheim: Consultancy; Novartis: Honoraria; Takeda: Honoraria; Oncimmune: Consultancy; Janssen: Consultancy. Cragg: BioInvent International: Consultancy, Research Funding; GSK: Research Funding; UCB: Research Funding; iTeos: Research Funding; Roche: Research Funding. Forconi: Novartis: Honoraria; Roche: Honoraria; Janssen: Consultancy, Honoraria, Speakers Bureau; AbbVie: Consultancy, Honoraria, Speakers Bureau; Gilead: Research Funding. Landesman: Karyopharm Therapeutics: Current Employment, Current equity holder in publicly-traded company. Blunt: Karyopharm Therapeutics: Research Funding.

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