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Comparable transforming growth factor beta-mediated immune suppression in ex vivo-expanded natural killer cells from cord blood and peripheral blood: implications for adoptive immunotherapy

Open AccessPublished:May 16, 2022DOI:https://doi.org/10.1016/j.jcyt.2022.04.001

      Abstract

      T cell-based therapies like genetically modified immune cells expressing chimeric antigen receptors have shown robust anti-cancer activity in vivo, especially in patients with blood cancers. However, extending this approach to an “off-the-shelf” setting can be challenging, as allogeneic T cells carry a significant risk of graft-versus-host disease (GVHD). By contrast, allogeneic natural killer (NK) cells recognize malignant cells without the need for prior antigen exposure and have been used safely in multiple cancer settings without the risk of GVHD. However, similar to T cells, NK cell function is negatively impacted by tumor-induced transforming growth factor beta (TGF-β) secretion, which is a ubiquitous and potent immunosuppressive mechanism employed by most malignancies. Allogeneic NK cells for adoptive immunotherapy can be sourced from peripheral blood (PB) or cord blood (CB), and the authors’ group and others have previously shown that ex vivo expansion and gene engineering can overcome CB-derived NK cells’ functional immaturity and poor cytolytic activity, including in the presence of exogenous TGF-β.  However, a direct comparison of the effects of TGF-β-mediated immune suppression on ex vivo-expanded CB- versus PB-derived NK cell therapy products has not previously been performed. Here the authors show that PB- and CB-derived NK cells have distinctive gene signatures that can be overcome by ex vivo expansion. Additionally, exposure to exogenous TGF-β results in an upregulation of inhibitory receptors on NK cells, a novel immunosuppressive mechanism not previously described. Finally, the authors provide functional and genetic evidence that both PB- and CB-derived NK cells are equivalently susceptible to TGF-β-mediated immune suppression. The authors believe these results provide important mechanistic insights to consider when using ex vivo-expanded, TGF-β-resistant PB- or CB-derived NK cells as novel immunotherapy agents for cancer.

      Keywords

      Abbreviation/Gene Annotation:

      Gene ID used (Gene Name), ITGB2 (integrin subunit beta 2(ITGB2)), PRF1 (perforin 1(PRF1)), PIK3CD (phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit delta(PIK3CD)), FASLG (Fas ligand(FASLG)), PIK3CB (phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit beta(PIK3CB)), ITGAL (integrin subunit alpha L(ITGAL)), TNF (tumor necrosis factor(TNF)), ACTB (actin beta(ACTB)), FCGR3A (Fc fragment of IgG receptor IIIa(FCGR3A)), FCGR3B (Fc fragment of IgG receptor IIIb(FCGR3B)), TNFSF10 (tumor necrosis factor superfamily member 10(TNFSF10)), RAC2 (ras-related C3 botulinum toxin substrate 2 (rho family, small GTP binding protein Rac2)(RAC2)), RAC3 (ras-related C3 botulinum toxin substrate 3 (rho family, small GTP binding protein Rac3)(RAC3)), RAC1 (ras-related C3 botulinum toxin substrate 1 (rho family, small GTP binding protein Rac1)(RAC1)), GUSB (glucuronidase beta(GUSB)), B2M (beta-2-microglobulin(B2M)), IFNAR2 (interferon alpha and beta receptor subunit 2(IFNAR2)), VAV3 (vav guanine nucleotide exchange factor 3(VAV3)), FCER1G (Fc fragment of IgE receptor Ig(FCER1G)), SYK (spleen associated tyrosine kinase(SYK)), RPL13A (ribosomal protein L13a(RPL13A)), VAV1 (vav guanine nucleotide exchange factor 1(VAV1)), VAV2 (vav guanine nucleotide exchange factor 2(VAV2)), NCR1 (natural cytotoxicity triggering receptor 1(NCR1)), ZAP70 (zeta chain of T cell receptor associated protein kinase 70(ZAP70)), NCR2 (natural cytotoxicity triggering receptor 2(NCR2)), NCR3 (natural cytotoxicity triggering receptor 3(NCR3)), TYROBP (TYRO protein tyrosine kinase binding protein(TYROBP)), IFNG (interferon gamma(IFNG)), PIK3CA (phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha(PIK3CA)), LCK (LCK proto-oncogene, Src family tyrosine kinase(LCK)), LCP2 (lymphocyte cytosolic protein 2(LCP2)), HPRT1 (hypoxanthine phosphoribosyltransferase 1(HPRT1)), RAF1 (Raf-1 proto-oncogene, serine/threonine kinase(RAF1)), PPIA (peptidylprolyl isomerase A(PPIA)), GAPDH (glyceraldehyde-3-phosphate dehydrogenase(GAPDH)), IFNAR1 (interferon alpha and beta receptor subunit 1(IFNAR1)), SHC2 (SHC adaptor protein 2(SHC2)), SHC1 (SHC adaptor protein 1(SHC1)), PIK3R3 (phosphoinositide-3-kinase regulatory subunit 3(PIK3R3)), PIK3R2 (phosphoinositide-3-kinase regulatory subunit 2(PIK3R2)), KIR2DL1 (killer cell immunoglobulin like receptor, two Ig domains and long cytoplasmic tail 1(KIR2DL1)), PIK3R1 (phosphoinositide-3-kinase regulatory subunit 1(PIK3R1)), KIR2DL2 (killer cell immunoglobulin like receptor, two Ig domains and long cytoplasmic tail 2(KIR2DL2)), KIR2DL3 (killer cell immunoglobulin like receptor, two Ig domains and long cytoplasmic tail 3(KIR2DL3)), KIR2DL4 (killer cell immunoglobulin like receptor, two Ig domains and long cytoplasmic tail 4(KIR2DL4)), PAK1 (p21 (RAC1) activated kinase 1(PAK1)), SH3BP2 (SH3 domain binding protein 2(SH3BP2)), PLCG2 (phospholipase C gamma 2(PLCG2)), PGK1 (phosphoglycerate kinase 1(PGK1)), MAPK1 (mitogen-activated protein kinase 1(MAPK1)), FYN (FYN proto-oncogene, Src family tyrosine kinase(FYN)), PLCG1 (phospholipase C gamma 1(PLCG1)), KLRC1 (killer cell lectin like receptor C1(KLRC1)), MAPK3 (mitogen-activated protein kinase 3(MAPK3)), KIR2DL5A (killer cell immunoglobulin like receptor, two Ig domains and long cytoplasmic tail 5A(KIR2DL5A)), KIR2DS1 (killer cell immunoglobulin like receptor, two Ig domains and short cytoplasmic tail 1(KIR2DS1)), KIR2DS2 (killer cell immunoglobulin like receptor, two Ig domains and short cytoplasmic tail 2(KIR2DS2)), KIR2DS3 (killer cell immunoglobulin like receptor, two Ig domains and short cytoplasmic tail 3(KIR2DS3)), SH2D1A (SH2 domain containing 1A(SH2D1A)), KIR2DS4 (killer cell immunoglobulin like receptor, two Ig domains and short cytoplasmic tail 4(KIR2DS4)), KIR2DS5 (killer cell immunoglobulin like receptor, two Ig domains and short cytoplasmic tail 5(KIR2DS5)), GZMB (granzyme B(GZMB)), TNFRSF10B (TNF receptor superfamily member 10b(TNFRSF10B)), NFATC2 (nuclear factor of activated T-cells 2(NFATC2)), PTPN11 (protein tyrosine phosphatase, non-receptor type 11(PTPN11)), KIR3DL1 (killer cell immunoglobulin like receptor, three Ig domains and long cytoplasmic tail 1(KIR3DL1)), NFATC1 (nuclear factor of activated T-cells 1(NFATC1)), KIR3DL2 (killer cell immunoglobulin like receptor, three Ig domains and long cytoplasmic tail 2(KIR3DL2)), FAS (Fas cell surface death receptor(FAS)), GRB2 (growth factor receptor bound protein 2(GRB2)), PTPN6 (protein tyrosine phosphatase, non-receptor type 6(PTPN6)), KLRD1 (killer cell lectin like receptor D1(KLRD1)), LAT (linker for activation of T-cells(LAT)), 2B4 (Natural Killer Cell Receptor 2B4)

      Introduction

      Allogeneic “off-the-shelf” natural killer (NK) cell therapeutics have seen increased use as adoptive immunotherapy for blood cancers and solid tumors [
      • Li D.
      • et al.
      Genetically engineered T cells for cancer immunotherapy.
      ,
      • Herrera L.
      • et al.
      Adult peripheral blood and umbilical cord blood NK cells are good sources for effective CAR therapy against CD19 positive leukemic cells.
      ,
      • Chaudhry K.
      • Dowlati E.
      • Bollard C.M.
      Chimeric antigen receptor-engineered natural killer cells: a promising cancer immunotherapy.
      ,
      • Yvon E.S.
      • et al.
      Cord blood natural killer cells expressing a dominant negative TGF-beta receptor: implications for adoptive immunotherapy for glioblastoma.
      ,
      • Pfefferle A.
      • Huntington N.D.
      You have got a fast car: chimeric antigen receptor NK cells in cancer therapy.
      ]. These therapies recognize malignant cells as a result of the absence of self (preventing engagement of inhibitory receptors) while avoiding non-self, healthy cells that do not express activating receptor ligands [
      • Basar R.
      • Daher M.
      • Rezvani K.
      Next-generation cell therapies: the emerging role of CAR-NK cells.
      ,
      • Sanchez C.E.
      • et al.
      NK cell adoptive immunotherapy of cancer: evaluating recognition strategies and overcoming limitations.
      ]. NK cell therapy using peripheral blood (PB) from healthy donors has shown promising results in clinical trials targeting leukemia after allogeneic stem cell transplantation [
      • Xing D.
      • et al.
      Cord blood natural killer cells exhibit impaired lytic immunological synapse formation that is reversed with IL-2 ex vivo expansion.
      ,
      • Miller J.S.
      • et al.
      Successful adoptive transfer and in vivo expansion of human haploidentical NK cells in patients with cancer.
      ,
      • Passweg J.R.
      • et al.
      Use of natural killer cells in hematopoetic stem cell transplantation.
      ,
      • Hsu K.C.
      • et al.
      Improved outcome in HLA-identical sibling hematopoietic stem-cell transplantation for acute myelogenous leukemia predicted by KIR and HLA genotypes.
      ] and is being actively explored in the blood cancer and solid tumor (including brain tumor) settings.
      Although the majority of clinical studies evaluating NK cell immunotherapy have used PB-derived NK cells, several alternative sources of NK cells exist, including human embryonic stem cells, induced pluripotent stem cells, artificial NK cell lines and umbilical cord blood (CB) [
      • Matsuo Y.
      • Drexler H.G.
      Immunoprofiling of cell lines derived from natural killer-cell and natural killer-like T-cell leukemia-lymphoma.
      ,
      • Woll P.S.
      • et al.
      Human embryonic stem cells differentiate into a homogeneous population of natural killer cells with potent in vivo antitumor activity.
      ,
      • Chouaib S.
      • et al.
      Improving the outcome of leukemia by natural killer cell-based immunotherapeutic strategies.
      ]. Most of these are limited, however. For example, obtaining NK cells from PB by apheresis or from bone marrow can be invasive, with potential risks to healthy donors [
      • Winters J.L.
      Complications of donor apheresis.
      ,
      • Miller J.P.
      • et al.
      Recovery and safety profiles of marrow and PBSC donors: experience of the National Marrow Donor Program.
      ,
      • Yuan S.
      • et al.
      Moderate and severe adverse events associated with apheresis donations: incidences and risk factors.
      ], and NK cell differentiation from human embryonic stem cells or induced pluripotent stem cells is largely experimental [
      • Knorr D.A.
      • et al.
      Clinical-scale derivation of natural killer cells from human pluripotent stem cells for cancer therapy.
      ,
      • Ni Z.
      • et al.
      Expression of chimeric receptor CD4ζ by natural killer cells derived from human pluripotent stem cells improves in vitro activity but does not enhance suppression of HIV infection in vivo.
      ], with a few therapies entering clinical trials [
      • Shankar K.
      • Capitini C.M.
      • Saha K.
      Genome engineering of induced pluripotent stem cells to manufacture natural killer cell therapies.
      ]. Artificial NK cell lines such as NK-92 [
      • Suck G.
      • et al.
      NK-92: an ‘off-the-shelf therapeutic’ for adoptive natural killer cell-based cancer immunotherapy.
      ,
      • Romanski A.
      • et al.
      CD19-CAR engineered NK-92 cells are sufficient to overcome NK cell resistance in B-cell malignancies.
      ,
      • Oelsner S.
      • et al.
      Continuously expanding CAR NK-92 cells display selective cytotoxicity against B-cell leukemia and lymphoma.
      ], KHYG-1 [
      • Kobayashi E.
      • et al.
      A chimeric antigen receptor for TRAIL-receptor 1 induces apoptosis in various types of tumor cells.
      ], NKL and NKG are derived from patients with NK cell lymphoma [
      • Gong J.H.
      • Maki G.
      • Klingemann H.G.
      Characterization of a human cell line (NK-92) with phenotypical and functional characteristics of activated natural killer cells.
      ] and thus possess the risk of potential tumor engraftment following infusion. By contrast, CB is more amenable to “off-the-shelf” approaches because it is readily available from hundreds of CB banks globally [
      • Rezvani K.
      • Rouce R.H.
      The application of natural killer cell immunotherapy for the treatment of cancer.
      ,
      • Dahlberg C.I.
      • et al.
      Natural killer cell-based therapies targeting cancer: possible strategies to gain and sustain anti-tumor activity.
      ,
      • Mehta R.S.
      • Shpall E.J.
      • Rezvani K.
      Cord blood as a source of natural killer cells.
      ].
      Although no direct comparison of PB- and CB-derived NK cell anti-tumor efficacy has been performed in the clinical setting, in vitro studies have identified several differences between these sources. CB contains a higher number of NK cell progenitors than PB, which potentially can differentiate into mature NK cells during ex vivo expansion [
      • Rutella S.
      • et al.
      Identification of a novel subpopulation of human cord blood CD34-CD133-CD7-CD45+lineage- cells capable of lymphoid/NK cell differentiation after in vitro exposure to IL-15.
      ,
      • Mehta R.S.
      • Shpall E.J.
      • Rezvani K.
      Cord blood as a source of natural killer cells.
      ]. However, resting CB-derived NK cells also show impaired ability to form F-actin immunological synapses (i.e., weaker conjugates with target cells) [
      • Xing D.
      • et al.
      Cord blood natural killer cells exhibit impaired lytic immunological synapse formation that is reversed with IL-2 ex vivo expansion.
      ,
      • Dalle J.-H.
      • et al.
      Characterization of cord blood natural killer cells: implications for transplantation and neonatal infections.
      ,
      • Tanaka H.
      • et al.
      Analysis of natural killer (NK) cell activity and adhesion molecules on NK cells from umbilical cord blood.
      ].  CB-derived NK cells also have a low expression level of adhesion molecules, which may contribute to this low NK cell activity [
      • Tanaka H.
      • et al.
      Analysis of natural killer (NK) cell activity and adhesion molecules on NK cells from umbilical cord blood.
      ]. Furthermore, CB-derived NK cells have lower percentages of tumor necrosis factor alpha-producing cells compared with PB-derived NK cells [
      • Krampera M.
      • et al.
      Intracellular cytokine profile of cord blood T-, and NK- cells and monocytes.
      ]. Tightly regulated receptor signaling between NK cells and susceptible tumor cells is essential for NK cell-mediated cytotoxicity [
      • Sanchez C.E.
      • et al.
      NK cell adoptive immunotherapy of cancer: evaluating recognition strategies and overcoming limitations.
      ,
      • Chan C.J.
      • Smyth M.J.
      • Martinet L.
      Molecular mechanisms of natural killer cell activation in response to cellular stress.
      ,
      • Vivier E.
      • et al.
      Targeting natural killer cells and natural killer T cells in cancer nature reviews.
      ]. CB-derived NK cells are phenotypically and functionally immature [
      • Alnabhan R.
      • Madrigal A.
      • Saudemont A.
      Differential activation of cord blood and peripheral blood natural killer cells by cytokines.
      ,
      • Shereck E.
      • et al.
      Immunophenotypic, cytotoxic, proteomic and genomic characterization of human cord blood vs. peripheral blood CD56(Dim) NK cells.
      ]. These limitations can be overcome by ex vivo activation of CB-derived NK cells by cytokines or K562-based artificial antigen presenting cells [
      • Xing D.
      • et al.
      Cord blood natural killer cells exhibit impaired lytic immunological synapse formation that is reversed with IL-2 ex vivo expansion.
      ,
      • Alnabhan R.
      • Madrigal A.
      • Saudemont A.
      Differential activation of cord blood and peripheral blood natural killer cells by cytokines.
      ,
      • Shaim H.
      • Yvon E.
      Cord blood: a promising source of allogeneic natural killer cells for immunotherapy.
      ]. This expansion process reliably generates clinically relevant doses of NK cells from a CB unit for adoptive immunotherapy [
      • Shah N.
      • et al.
      Antigen presenting cell-mediated expansion of human umbilical cord blood yields log-scale expansion of natural killer cells with anti-myeloma activity.
      ]. Expanded CB-derived NK cells show an increased level of eomesodermin and T-bet [
      • Liu E.
      • et al.
      Cord blood NK cells engineered to express IL-15 and a CD19-targeted CAR show long-term persistence and potent antitumor activity.
      ]. These two transcription factors play essential roles in the ability of NK cells to develop and acquire effector functions [
      • Werneck M.B.
      • et al.
      T-bet plays a key role in NK-mediated control of melanoma metastatic disease.
      ,
      • Townsend M.J.
      • et al.
      T-bet regulates the terminal maturation and homeostasis of NK and Valpha14i NKT cells.
      ,
      • Pearce E.L.
      • et al.
      Control of effector CD8+ T cell function by the transcription factor Eomesodermin.
      ]. However, thus far the authors have not seen studies that have evaluated the impact of immune suppression on expanded NK cells from CB versus PB sources. Transforming growth factor beta (TGF-β) is a potent immunosuppressive cytokine released abundantly in the tumor microenvironment with inhibitory effects on NK cell functions such as downregulation of interferon gamma (IFN-γ) production and expression of activating receptors like NKG2D [
      • Yvon E.S.
      • et al.
      Cord blood natural killer cells expressing a dominant negative TGF-beta receptor: implications for adoptive immunotherapy for glioblastoma.
      ,
      • Dasgupta S.
      • et al.
      Inhibition of NK cell activity through TGF-beta 1 by down-regulation of NKG2D in a murine model of head and neck cancer.
      ,
      • Meadows S.K.
      • et al.
      Human NK cell IFN-gamma production is regulated by endogenous TGF-beta.
      ,
      • Lee H.M.
      • Kim K.S.
      • Kim J.
      A comparative study of the effects of inhibitory cytokines on human natural killer cells and the mechanistic features of transforming growth factor-beta.
      ]. CB-derived NK cells may potentially be more susceptible to the effects of TGF-β, as this cytokine is also critical in maintaining tolerance in the placental environment from which these cells are derived [
      • Yang D.
      • et al.
      Role of transforming growth factor-β1 in regulating fetal-maternal immune tolerance in normal and pathological pregnancy.
      ].
      Here the authors investigate CB- versus PB-derived NK cells derived from healthy individuals with and without ex vivo expansion and evaluate how these NK cell products respond to TGF-β-mediated immune suppression. The authors hypothesized that TGF-β-mediated immune suppression in ex vivo-expanded CB-derived NK cells would be comparable to PB-derived NK cells in phenotype, functional activity and gene expression signature.

      Methods

      Cell source and cell lines

      Umbilical CB and PB were obtained under Children's National Hospital institutional review board-approved protocols Pro0004033, Pro00003896, Pro00009374 and Pro00003869. PB- and CB-derived mononuclear cells (MNCs) were harvested by density gradient separation with Lymphoprep (STEMCELL Technologies, Vancouver, Canada). K562 cell line (American Type Culture Collection, Manassas, VA, USA) was cultured with Roswell Park Memorial Institute medium supplemented with 10% fetal bovine serum (FBS), 1% penicillin–streptomycin and 1% GlutaMAX (Thermo Fisher Scientific, Waltham, MA, USA). U87 MG cells were purchased from American Type Culture Collection and cultured in Dulbecco's Modified Eagle's Medium:F12 medium (1:1) with 10% FBS and 1% GlutaMAX. All cell lines were tested regularly for mycoplasma contamination.

      RNA sequencing analysis

      Total RNA from frozen MNCs was isolated using an miRNeasy Mini Kit (QIAGEN, Hilden, Germany). A KAPA RNA HyperPrep Kit with RiboErase (Roche Sequencing Solutions, Pleasanton, CA, USA) was used for library preparation and sequenced on a NovaSeq 6000 (Illumina, San Diego, CA, USA) using 100 paired-end sequencing. Quality control for raw reads was performed with FastQC [
      • Subramanian A.
      • et al.
      Gene set enrichment analysis: a knowledge-based approach for interpreting genome-wide expression profiles.
      ]. Spliced alignment of the reads to the reference genome was done with STAR followed by a second round of quality control using RSeQC [
      • Wang L.
      • Wang S.
      • Li W.
      RSeQC: quality control of RNA-seq experiments.
      ]. Samples were aligned to the human genome using reference build GRCh38.p7 and the corresponding GENCODE annotation version 25 (March 2016). Mapped reads were quantified at the gene level as a raw counts matrix using featureCounts from Subread [
      • Liao Y.
      • Smyth G.K.
      • Shi W.
      The Subread aligner: fast, accurate and scalable read mapping by seed-and-vote.
      ] with fracOverlap 1 (only entire reads overlapping to annotation feature are counted). Raw feature counts were normalized, and differential expression analysis was carried out using DESeq2 [
      • Love M.I.
      • Huber W.
      • Anders S.
      Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2.
      ]. Differential expression rank order was used for subsequent gene set enrichment analysis performed using the clusterProfiler package in R. Gene set variation analysis was applied on select gene sets using the gene set variation analysis package in R. Gene sets queried included the hallmark and canonical pathways and Gene Ontology biological processes collections available through the Molecular Signatures Database [
      • Subramanian A.
      • et al.
      Gene set enrichment analysis: a knowledge-based approach for interpreting genome-wide expression profiles.
      ].

      Expansion of NK cells

      NK cells were isolated by negative selection with the EasySep Human NK Cell Isolation Kit (STEMCELL Technologies). After 24 h of activation with 10 ng/mL human IL-15 (R&D Systems, Minneapolis, MN, USA), NK cells were stimulated with unmodified K562 feeder cells previously irradiated at 200 Gy. Irradiated K562 cells were cultured with NK cells at a 2:1 K562:NK cell ratio. NK cells were expanded in stem cell growth medium supplemented with 200 IU/mL human IL-2, 10 ng/mL human IL-15, 10% FBS and 1% GlutaMAX. NK cells were stimulated again on day 12 of culture at a 2:1 K562:NK cell ratio. Sample sizes (number of donor-derived lines) used for each experiment are specified in Figure 1, Figure 2, Figure 3, Figure 4, Figure 5, Figure 6.
      Fig 1
      Figure 1Transcriptional profiling of MNCs from PB and CB. (A) Heat map depicting all differentially expressed genes identified between CB- and PB-derived MNCs. Dendrograms show the hierarchical clustering (Pearson distance) between samples. CB donors are highlighted by blue and PB donors are denoted by red (n = 5 per group). (B) Heat map of candidate gene expression measured by RT-qPCR in ex vivo-expanded NK cells from CB and PB (blue representing downregulation, red representing upregulation relative to the housekeeping genes). Cells were expanded ex vivo as described. All experiments were undertaken in triplicate. CB donors are highlighted by blue and PB donors are denoted by red. Pathways are categorized as NK cell effector function pathways (orange), including DAP signaling and calcium signaling. NK cell inhibition pathway genes are indicated in blue. NK cell activation pathway genes and MAPK pathways are shown in green. Cytokine and lytic enzyme pathway genes are indicated in pink (n = 5). (C) CB- and PB-derived NK cell phenotype, including NK cell activation, maturation and exhaustion receptors. CD3– and CD56+ cells were gated to quantify NK cell population and for further NK receptor analysis. Ex vivo-expanded CB-derived NK cells (blue bars) lacked any phenotypic evidence of exhaustion and maintained a phenotype similar to that of PB cells (red bars). Each dot represents individual donor and error bars represent standard deviation (n = 8). (D,E) Anti-tumor function of PB- and CB-derived NK cells as measured by luciferase-based cytotoxicity assay against U87 MG cells (n = 6). CB-derived NK cells (blue line) were as efficient as PB-derived NK cells (red line) in killing U87 MG targets at different E:T ratios (x-axis). (D) PB- and CB-derived NK cell 4-h cytotoxicity assay (n = 6) target lysis. (E) PB- and CB-derived NK cell 72-h cytotoxicity assay (n = 4) target lysis. Error bars represent standard deviation. RT-qPCR, real-time qPCR. (Color version of figure is available online).
      Fig 1
      Figure 1Transcriptional profiling of MNCs from PB and CB. (A) Heat map depicting all differentially expressed genes identified between CB- and PB-derived MNCs. Dendrograms show the hierarchical clustering (Pearson distance) between samples. CB donors are highlighted by blue and PB donors are denoted by red (n = 5 per group). (B) Heat map of candidate gene expression measured by RT-qPCR in ex vivo-expanded NK cells from CB and PB (blue representing downregulation, red representing upregulation relative to the housekeeping genes). Cells were expanded ex vivo as described. All experiments were undertaken in triplicate. CB donors are highlighted by blue and PB donors are denoted by red. Pathways are categorized as NK cell effector function pathways (orange), including DAP signaling and calcium signaling. NK cell inhibition pathway genes are indicated in blue. NK cell activation pathway genes and MAPK pathways are shown in green. Cytokine and lytic enzyme pathway genes are indicated in pink (n = 5). (C) CB- and PB-derived NK cell phenotype, including NK cell activation, maturation and exhaustion receptors. CD3– and CD56+ cells were gated to quantify NK cell population and for further NK receptor analysis. Ex vivo-expanded CB-derived NK cells (blue bars) lacked any phenotypic evidence of exhaustion and maintained a phenotype similar to that of PB cells (red bars). Each dot represents individual donor and error bars represent standard deviation (n = 8). (D,E) Anti-tumor function of PB- and CB-derived NK cells as measured by luciferase-based cytotoxicity assay against U87 MG cells (n = 6). CB-derived NK cells (blue line) were as efficient as PB-derived NK cells (red line) in killing U87 MG targets at different E:T ratios (x-axis). (D) PB- and CB-derived NK cell 4-h cytotoxicity assay (n = 6) target lysis. (E) PB- and CB-derived NK cell 72-h cytotoxicity assay (n = 4) target lysis. Error bars represent standard deviation. RT-qPCR, real-time qPCR. (Color version of figure is available online).
      Fig 1
      Figure 1Transcriptional profiling of MNCs from PB and CB. (A) Heat map depicting all differentially expressed genes identified between CB- and PB-derived MNCs. Dendrograms show the hierarchical clustering (Pearson distance) between samples. CB donors are highlighted by blue and PB donors are denoted by red (n = 5 per group). (B) Heat map of candidate gene expression measured by RT-qPCR in ex vivo-expanded NK cells from CB and PB (blue representing downregulation, red representing upregulation relative to the housekeeping genes). Cells were expanded ex vivo as described. All experiments were undertaken in triplicate. CB donors are highlighted by blue and PB donors are denoted by red. Pathways are categorized as NK cell effector function pathways (orange), including DAP signaling and calcium signaling. NK cell inhibition pathway genes are indicated in blue. NK cell activation pathway genes and MAPK pathways are shown in green. Cytokine and lytic enzyme pathway genes are indicated in pink (n = 5). (C) CB- and PB-derived NK cell phenotype, including NK cell activation, maturation and exhaustion receptors. CD3– and CD56+ cells were gated to quantify NK cell population and for further NK receptor analysis. Ex vivo-expanded CB-derived NK cells (blue bars) lacked any phenotypic evidence of exhaustion and maintained a phenotype similar to that of PB cells (red bars). Each dot represents individual donor and error bars represent standard deviation (n = 8). (D,E) Anti-tumor function of PB- and CB-derived NK cells as measured by luciferase-based cytotoxicity assay against U87 MG cells (n = 6). CB-derived NK cells (blue line) were as efficient as PB-derived NK cells (red line) in killing U87 MG targets at different E:T ratios (x-axis). (D) PB- and CB-derived NK cell 4-h cytotoxicity assay (n = 6) target lysis. (E) PB- and CB-derived NK cell 72-h cytotoxicity assay (n = 4) target lysis. Error bars represent standard deviation. RT-qPCR, real-time qPCR. (Color version of figure is available online).
      Fig 2
      Figure 2Signaling pathways in NK cells. (A) MFI of pSMAD2. CB- and PB-derived NK cells were treated with 5 ng/mL TGF-β for 1 h before assaying for differences in pSMAD2, as measured by Luminex (n = 5). Red bars represent phosphorylated SAMD2 in NK cells in the presence of TGF-β and black bars represent the absence of TGF-β. Each dot represents individual donor and error bars represent standard deviation. (B) MFI of pSMAD3. CB- and PB-derived NK cells were treated with 5 ng/mL TGF-β for 1 h before assaying for differences in pSMAD3, as measured by Luminex (n = 5). Red bars represent phosphorylated SMAD3 in NK cells in the presence of TGF-β and black bars represent the absence of TGF-β. Two-way ANOVA was used to determine significance between the groups.(C) pSMAD2/3 upregulation kinetics. CB- and PB-derived NK cells were treated with 5 ng/mL TGF-β for 1 min, 5 min, 10 min and 30 min and assessed by phosphoflow for pSMAD2/3. Each dot represents individual donor and error bars represent standard deviation. Ratio-paired t-tests were used to determine statistically significant differences. Black bars represent PB-derived NK cells and Red bars represent CB-derived NK cells.(D) Representative pSMAD2/3 expression on CB- and PB-derived NK cells after incubation with TGF-β at different time points. Ctrl sample is NK cells incubated without TGF-β. *P < 0.05. ANOVA, analysis of variance; Ctrl, control; MFI, mean fluorescence intensity. (Color version of figure is available online).
      Fig 3
      Figure 3Gene expression alterations associated with exogenous TGF-β treatment. PB- and CB-derived NK cells were cultured in the absence and presence of TGF-β for 5 days. Cells were then analyzed by qPCR to determine the change in NK cell effector function in response to TGF-β treatment. (A–D) Expression results are presented as a hierarchical cluster heat map. Each row denotes a parameter and each column denotes a cell culture condition as indicated. The color scale indicates relative protein abundance as determined by fold change. Dendrograms denote the Euclidean distances between clustered conditions. (A) NK cell activation pathway genes. (B) NK cell inhibition pathway genes. (C) Cytokine and lytic enzyme pathway genes. (D) NK cell effector function pathways. (E–H) Mean gene expression change in NK cells in the presence of TGF-β relative to NK cells in the absence of TGF-β is shown by fold change (y-axis). (E) NK cell effector function pathways. (F) NK cell inhibition pathway genes. (G) Cytokine and lytic enzyme pathway genes. (H) NK cell activation pathway genes. PB-derived NK cells are represented by black boxes, CB-derived NK cells are represented by red boxes and whisker extends from minimum to maximum value. Each dot represents individual donor. (Color version of figure is available online).
      Fig 3
      Figure 3Gene expression alterations associated with exogenous TGF-β treatment. PB- and CB-derived NK cells were cultured in the absence and presence of TGF-β for 5 days. Cells were then analyzed by qPCR to determine the change in NK cell effector function in response to TGF-β treatment. (A–D) Expression results are presented as a hierarchical cluster heat map. Each row denotes a parameter and each column denotes a cell culture condition as indicated. The color scale indicates relative protein abundance as determined by fold change. Dendrograms denote the Euclidean distances between clustered conditions. (A) NK cell activation pathway genes. (B) NK cell inhibition pathway genes. (C) Cytokine and lytic enzyme pathway genes. (D) NK cell effector function pathways. (E–H) Mean gene expression change in NK cells in the presence of TGF-β relative to NK cells in the absence of TGF-β is shown by fold change (y-axis). (E) NK cell effector function pathways. (F) NK cell inhibition pathway genes. (G) Cytokine and lytic enzyme pathway genes. (H) NK cell activation pathway genes. PB-derived NK cells are represented by black boxes, CB-derived NK cells are represented by red boxes and whisker extends from minimum to maximum value. Each dot represents individual donor. (Color version of figure is available online).
      Fig 3
      Figure 3Gene expression alterations associated with exogenous TGF-β treatment. PB- and CB-derived NK cells were cultured in the absence and presence of TGF-β for 5 days. Cells were then analyzed by qPCR to determine the change in NK cell effector function in response to TGF-β treatment. (A–D) Expression results are presented as a hierarchical cluster heat map. Each row denotes a parameter and each column denotes a cell culture condition as indicated. The color scale indicates relative protein abundance as determined by fold change. Dendrograms denote the Euclidean distances between clustered conditions. (A) NK cell activation pathway genes. (B) NK cell inhibition pathway genes. (C) Cytokine and lytic enzyme pathway genes. (D) NK cell effector function pathways. (E–H) Mean gene expression change in NK cells in the presence of TGF-β relative to NK cells in the absence of TGF-β is shown by fold change (y-axis). (E) NK cell effector function pathways. (F) NK cell inhibition pathway genes. (G) Cytokine and lytic enzyme pathway genes. (H) NK cell activation pathway genes. PB-derived NK cells are represented by black boxes, CB-derived NK cells are represented by red boxes and whisker extends from minimum to maximum value. Each dot represents individual donor. (Color version of figure is available online).
      Fig 3
      Figure 3Gene expression alterations associated with exogenous TGF-β treatment. PB- and CB-derived NK cells were cultured in the absence and presence of TGF-β for 5 days. Cells were then analyzed by qPCR to determine the change in NK cell effector function in response to TGF-β treatment. (A–D) Expression results are presented as a hierarchical cluster heat map. Each row denotes a parameter and each column denotes a cell culture condition as indicated. The color scale indicates relative protein abundance as determined by fold change. Dendrograms denote the Euclidean distances between clustered conditions. (A) NK cell activation pathway genes. (B) NK cell inhibition pathway genes. (C) Cytokine and lytic enzyme pathway genes. (D) NK cell effector function pathways. (E–H) Mean gene expression change in NK cells in the presence of TGF-β relative to NK cells in the absence of TGF-β is shown by fold change (y-axis). (E) NK cell effector function pathways. (F) NK cell inhibition pathway genes. (G) Cytokine and lytic enzyme pathway genes. (H) NK cell activation pathway genes. PB-derived NK cells are represented by black boxes, CB-derived NK cells are represented by red boxes and whisker extends from minimum to maximum value. Each dot represents individual donor. (Color version of figure is available online).
      Fig 3
      Figure 3Gene expression alterations associated with exogenous TGF-β treatment. PB- and CB-derived NK cells were cultured in the absence and presence of TGF-β for 5 days. Cells were then analyzed by qPCR to determine the change in NK cell effector function in response to TGF-β treatment. (A–D) Expression results are presented as a hierarchical cluster heat map. Each row denotes a parameter and each column denotes a cell culture condition as indicated. The color scale indicates relative protein abundance as determined by fold change. Dendrograms denote the Euclidean distances between clustered conditions. (A) NK cell activation pathway genes. (B) NK cell inhibition pathway genes. (C) Cytokine and lytic enzyme pathway genes. (D) NK cell effector function pathways. (E–H) Mean gene expression change in NK cells in the presence of TGF-β relative to NK cells in the absence of TGF-β is shown by fold change (y-axis). (E) NK cell effector function pathways. (F) NK cell inhibition pathway genes. (G) Cytokine and lytic enzyme pathway genes. (H) NK cell activation pathway genes. PB-derived NK cells are represented by black boxes, CB-derived NK cells are represented by red boxes and whisker extends from minimum to maximum value. Each dot represents individual donor. (Color version of figure is available online).
      Fig 4
      Figure 4Functional loss of ex vivo-expanded PB- and CB-derived NK cells in the presence of exogenous TGF-β. (A) Mean fold expansion of PB- and CB-derived NK cells in the presence and absence of TGF-β (n = 6). (B–D) Expression of NKG2D, DNAM1 and NKp30 was evaluated by flow cytometry after 5 days of TGF-β treatment (n = 5). (E–G) Cytokine secretion by PB- and CB-derived NK cells after exposure to TGF-β (n = 4). (E) Perforin, (F) granzyme and (G) IFN-γ secretion was detected by multiplex after 5-day exposure to TGF-β (n = 4). Red bars represent NK cells in the presence of TGF-β and black bars represent NK cells in the absence of TGF-β. Each dot represents individual donor and error bars represent standard deviation. Two-way ANOVA was used to determine statistically significant differences. *P < 0.05. ANOVA, analysis of variance. (Color version of figure is available online).
      Fig 5
      Figure 5Exogenous TGF-β decreases the cytolytic activity of PB- and CB-derived NK cells equivalently. Ex vivo-expanded PB- and CB-derived NK cells were cultured in the presence or absence of 5 ng/mL TGF-β for an additional 5 days. Specific lysis of the targets was evaluated using luciferase-based cytotoxicity assay against U87 MG cells (n = 6). Mean cytolytic ability (y-axis) of NK cells cultured with TGF-β for 5 days (red line) compared with control NK cells (black line) at different E:T ratios (x-axis). Data are presented as target cell lysis. Error bars represent standard deviation. (A) Representative PB donor target lysis measured at 4 h. (B) Representative CB donor target lysis measured at 4 h. (C) Decrease in target lysis by PB-derived (red bars) and CB-derived (black bars) NK cells measured at 4 h. Each dot represents individual donor and error bars represent standard deviation. Two-way ANOVA was used to determine statistically significant differences. *P < 0.05. ANOVA, analysis of variance. (Color version of figure is available online).
      Fig 6
      Figure 6Characterization of PB- and CB-derived NK cell downregulation of mTOR-regulated proteins in the presence of exogenous TGF-β. Expression of (A) CD71 and (B) CD122 was evaluated on PB- and CB-derived NK cells by flow cytometry after 5-day exposure to TGF-β by flow cytometry (n = 5). Red bars represent NK cells in the presence of TGF-β and black bars represent NK cells in the absence of TGF-β. Each dot represents individual donor and error bars represent standard deviation. Two-way ANOVA was used to determine statistically significant differences. *P < 0.05. ANOVA, analysis of variance. (Color version of figure is available online).

      Phenotypic assessment of expanded NK cells

      NK cells were harvested from 21- to 28-day cultures, washed with fluorescence-activated cell sorting buffer and incubated with human FcR blocking reagent for 5 min followed by antibody staining for 15 min at room temperature in the dark. NK cell phenotypes were assessed by flow cytometry using antibodies specific to human CD3 (PE-cy7 and Bv421), CD16 (APC-cy7 and BV650), CD25 (BV421), CD56 (PerCp/Cyanine5.5 and allophycocyanin [APC]), CD57 (fluorescein isothiocyanate), CD69 (APC-cy7 and BV650), NKp30 (APC), NKp46 (phycoerythrin [PE]), NKp44 (fluorescein isothiocyanate), NKG2D (BV421), TGF-βRII (APC and PE), TIM-3 (PE) and PD-1 (APC) from BD Biosciences (Franklin Lakes, NJ, USA), Thermo Fisher Scientific and BioLegend (San Diego, CA, USA). To distinguish between live and dead populations, samples were stained with fixable viability dyes. Next, samples were fixed using a BD Cytofix/Cytoperm Plus Fixation/Permeabilization Kit (BD Biosciences). Samples were acquired with CytoFLEX (Beckman Coulter, Brea, CA, USA). For each sample, a minimum of 10, 000 events were acquired, and data were analyzed using FlowJo 10 (Becton, Dickinson and Company, Franklin Lakes, NJ, USA).

      Signaling assessment of NK cells after exposure to TGF-β

      PB- and CB-derived NK cell samples (2 × 106 cells) were starved of serum and cytokines for 4 h and then treated with 5 ng/mL TGF-β as previously described [
      • Yvon E.S.
      • et al.
      Cord blood natural killer cells expressing a dominant negative TGF-beta receptor: implications for adoptive immunotherapy for glioblastoma.
      ]. TGF-β Treated and untreated cells were lysed, and collected supernatant was assayed using the MILLIPLEX MAP TGF-β kit (EMD Millipore, Burlington, MA, USA) according to the manufacturer's instructions, looking at levels of pSMAD2 and pSMAD3.

      Phenotypic and functional assessment of NK cells after exposure to TGF-β

      NK cells were cultured, and 5 ng/mL TGF-β (activated with 4 mmol/L hydrogen chloride) was added on day 1 and day 3. After 5 days of treatment, NK cells were examined by flow cytometry or cytotoxicity assays [
      • Yvon E.S.
      • et al.
      Cord blood natural killer cells expressing a dominant negative TGF-beta receptor: implications for adoptive immunotherapy for glioblastoma.
      ]. Cytolytic ability of the NK cell products after exposure to TGF-β was determined using luciferase-based biophotonic cytolytic assay [
      • Brown C.E.
      • et al.
      Biophotonic cytotoxicity assay for high-throughput screening of cytolytic killing.
      ,
      • Karimi M.A.
      • et al.
      Measuring cytotoxicity by bioluminescence imaging outperforms the standard chromium-51 release assay.
      ]. Briefly, NK cells were co-cultured with luciferase-expressing, TGF-β-secreting U87 MG cells at different Effector to target ratios. After 4 or 72 h of incubation, we added luciferin to each well at a final concentration of 75 μg/mL. The plate was dark-adapted for 5–10 min, and luminescence was read using a plate reader.

      Quantitative polymerase chain reaction

      Total RNA was extracted using a Monarch Total RNA Miniprep Kit (New England Biolabs, Ipswich, MA, USA). RNA was reverse-transcribed using an AzuraFlex cDNA Synthesis Kit (Azura Genomics, Raynham, MA, USA). Real-time quantitative polymerase chain reaction (qPCR) was performed using AzuraView GreenFast qPCR Blue Mix low 6-carboxy-X-rhodamine master mix (Azura Genomics) on QuantStudio II (Thermo Fisher Scientific). The relative expression levels of target genes were measured and normalized against levels of housekeeping genes ACTB, B2M, GAPDH, GUSB, HPRT1, PGK1, PPIA and RPL13A. Fold difference (as relative messenger RNA [mRNA] expression) was calculated by the delta Ct method [
      • Livak K.J.
      • Schmittgen T.D.
      Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) method.
      ]. Human NK Cell Mediated Toxicity Primer Library was purchased from Real Time Primers (Elkins Park, PA, USA). To calculate the significance of gene expression, the authors performed multiple t-tests, assuming that the housekeeping genes used for normalization did not vary between PB- and CB-derived NK cells. The relative change in gene expression was calculated as Δpb = PB – PBTGF-β and Δcb = CB – CBTGF-β.

      Quantification and statistical analysis

      Two-way analysis of variance was used to determine statistically significant differences between donor sources unless otherwise noted. Holm–Sidak test adjusted P < 0.05 indicated a significant difference. Graph generation and statistical analyses were performed using Prism software (GraphPad Software, San Diego, CA, USA).

      Results

      Ex vivo expansion resulted in a similar surface marker phenotype for CB- and PB-derived NK cells. Following bulk RNA sequencing experiments using MNCs from PB of five healthy donors and five umbilical CB samples (obtained under Children's National Hospital institutional review board-approved protocols Pro0004033, Pro00003896, Pro00009374 and Pro00003869), the authors initially observed that CB-derived MNCs had gene expression profiles that were distinct from those of PB-derived MNCs (Figure 1A). Analysis of global transcriptional profiles demonstrated that the two sources had a broad difference in gene expression of MNCs (differentially expressed genes, 3422 , false discovery rate, <0.05, fold change, >2), with 1593 upregulated and 2200 downregulated in PB-derived MNCs compared with CB-derived MNCs (see supplementary Figure 1A–D), similar to what the authors’ and others have previously observed [
      • Xing D.
      • et al.
      Cord blood natural killer cells exhibit impaired lytic immunological synapse formation that is reversed with IL-2 ex vivo expansion.
      ,
      • Shereck E.
      • et al.
      Immunophenotypic, cytotoxic, proteomic and genomic characterization of human cord blood vs. peripheral blood CD56Dim NK cells.
      ]. NK cell immune population-related pathways were significantly (P < 0.05, n = 5) downregulated in CB (see supplementary Figure 1E). The authors found that the NK cell percentage was low in both CB and PB (10–20%) [
      • Luevano M.
      • et al.
      The unique profile of cord blood natural killer cells balances incomplete maturation and effective killing function upon activation.
      ]. Therefore, to ensure a more accurate resolution between two donors, the authors also compared NK cell mRNA expression following CD56+ cell selection from CB- and PB-derived MNCs using qPCR. The authors’ qPCR panel consisted of 64 genes, including NK cell activating receptors, inhibitory receptors, cytokines and genes associated with effector function. Even though the authors observed minor clustering within the donor source, the differential expression of these genes was not significant (P > 0.05) (see supplementary Figure 1F). The authors posited that the difference in results between the two methods might be because qPCR was not as sensitive as RNA sequencing or that our “n” for qPCR (n = 3) was too small to draw a significant conclusion.
      Following ex vivo expansion, these differential gene signatures remained mostly intact (Fig. 1B). By contrast, as observed in the authors’ previous study, the phenotypic signatures became more aligned between CB- and PB-derived NK cells post-expansion (Fig. 1C) [
      • Xing D.
      • et al.
      Cord blood natural killer cells exhibit impaired lytic immunological synapse formation that is reversed with IL-2 ex vivo expansion.
      ]. Expanded NK cells showed equally high purity post-expansion, at 78.98% for CB donors and 86.6% for PB donors (P > 0.05, n >5) (Fig. 1C). Staining for natural cytotoxicity receptors NKG2D, NKp44, NKp44 and NKp30 showed no significant difference in expression between CB- and PB-derived NK cells (NKp44, 51.91% versus 47.26%, P > 0.05, NKp30, 60.14% versus 58.49%, P > 0.05, NKG2D, 92.96% versus 94.41%, P > 0.05, NKp46, 61.51% versus 59.29%, P > 0.05). Similarly, the authors noted no impairment in the expression of NK cell surface markers CD69, CD16, CD25, TIM3 or PD1 (P > 0.05). CB- and PB-derived NK cells showed similar cytolysis of U87 MG cells at different effector-to-target (E:T) ratios for both in a 4-h luciferase-based cytotoxicity assay (10:1, 51.93% versus 52.21%, P > 0.05, 5:1, 31.64% versus 33.60%, P > 0.05) (Fig. 1D) and a 72-h luciferase-based cytotoxicity assay (10:1, 100.4% versus 100.3%, P > 0.05, 5:1, 100.2% versus 100.2%, P > 0.05, 1:1, 93.2% versus 93.5%, P > 0.05) (Fig. 1E).
      Ex vivo-expanded NK cells from CB and PB had comparable signaling following TGF-β treatment. To determine whether CB- and PB-derived NK cells responded to TGF-β in a comparable manner, the authors evaluated TGF-β pathway activation in PB- and CB-derived NK cells after they were treated with 5 ng/mL TGF-β for 60 min. As shown in Fig. 2, pSMAD2 and pSAMD3 were comparably upregulated in both PB- and CB-derived NK cells in the presence of exogenous TGF-β (P < 0.05, n = 5). For PB-derived NK cells, the authors observed an increase from a mean of 62.7 ± 22.02 to 3246.38 ± 2046.23 (P < 0.05, n = 5) for pSMAD2 and an increase from a mean of 16.02 ± 2.95 to 221.26 ± 91.959 (P < 0.05, n = 5) for pSMAD3, as measured by ratio-paired t-tests. The results also showed upregulation of both pSMAD2 and pSMAD3 in CB-derived NK cells in the presence of TGF-β, from a mean of 55.78 ± 22.1 to 983.48 ± 97.21 for pSMAD2 and a mean of 18.36 ± 8.05 to 112.24 ± 82.3 for pSMAD3 (Fig. 2A,B). However, only upregulation of pSMAD3 was borderline significant in CB-derived NK cells in the presence of TGF-β, as measured by ratio-paired t-tests (P = 0.057). Hence, these studies suggested that the addition of exogenous TGF-β elicited a comparable increase in SMAD2 and SMAD3 phosphorylation in both CB- and PB-derived NK cells, though upregulation in CB was not statistically significant.
      The authors also performed a pSMAD2/3 upregulation kinetics study at different incubation times (1 min, 5 min, 10 min and 30 min) with TGF-β to evaluate for any differences between PB- and CB-derived NK cell response (Fig. 2C,D). Mean fluorescence intensity of pSMAD2/3 for PB-derived NK cells increased from 537.33 ± 21.39 (without TGF-β) to 678.67 ± 26.72 at 1 min and 875 ± 97.23 at 5 min (P < 0.05, n = 3), maintaining at 865.33 ± 152.13 by 10-min (P < 0.05, n = 3) incubation with TGF-β. By 30-min TGF-β exposure, pSMAD2/3 expression dropped to 711 ± 84.59 (Fig. 2C). CB-derived NK cells showed the same trend. Specifically, pSMAD2/3 mean fluorescence intensity increased from 573.33 ± 7.51 (without TGF-β) to 741.67 ± 97.37 at 1 min and 861.33 ± 41.02 at 5 min (P < 0.05, n = 3) and maintained at 843.33 ± 66.03 by 10 min (P < 0.05, n = 3). Moreover, pSMAD2/3 expression dropped in CB-derived NK cells to 714.67 ± 23.96 by 30 min. However, no significant differences were observed in pSMAD2/3 upregulation at different time intervals (P > 0.05, n = 3) (Fig. 2D) irrespective of the donor source.
      Ex vivo-expanded NK cells from CB and PB had similar gene expression signatures following TGF-β treatment. The authors evaluated whether the addition of TGF-β resulted in different gene expression signatures in CB- versus PB-derived NK cells. The unbiased clustering of relative changes (log2 fold change) in individual gene expression (log2 fold change) in the presence of TGF-β revealed that ex vivo-expanded PB- and CB-derived NK cells behaved similarly in response to the cytokine (Fig. 3A–D). The authors did see a minor clustering of donors according to their source of inhibitory pathway group (Fig. 3B) as well as cytokine and lytic enzyme pathway genes (Fig. 3C); however, analysis of the changes in gene expression between PB and CB donor sources was not found to be significant (P < 0.05).
      The authors observed a trend of downregulation of genes associated with NK cell activation, specifically natural cytotoxicity triggering receptor (NCR) 3 and integrin subunit beta 2, which were significantly downregulated for both CB- and PB-derived NK cells (P < 0.05) (Fig. 3E; also see supplementary Figure 3A). Furthermore, the authors observed a trend of upregulation of inhibitory genes (Fig. 3B,F) for both CB- and PB-derived NK cells upon exposure to TGF-β, including significant induction of growth factor receptor-bound protein 2 and protein tyrosine phosphatase non-receptor type 6 (P < 0.05, n = 5) (Fig. 3F). The upregulation of inhibitory pathway genes and downregulation of activating pathway genes reflected the loss of NK cell cytotoxicity frequently observed in the presence of exogenous TGF-β.
      The cytokine pathway gene interferon alpha and beta receptor subunit (IFNAR) 1 demonstrated upregulation in response to exposure to TGF-β irrespective of donor source (Fig. 3C). Granzyme B, tumor necrosis factor and IFN-γ expression change on exposure to exogenous TGF-β was inconclusive because of donor-specific variation within the current sample size (n = 5). The lytic enzyme perforin was downregulated in both PB- and CB-derived NK cells in the presence of TGF-β (Fig. 3G); however, it was only significantly downregulated for PB-derived NK cells. Genes associated with NK cell effector functions showed a mixed response and no clustering based on donor source (Fig. 3H). The majority of genes in this group showed a trend of upregulation—for example, LCK proto-oncogene Src family tyrosine kinase, lymphocyte cytosolic protein 2 (LCP2), mitogen-activated protein kinase 1 (MAPK1), nuclear factor of activated T cells (NFATC) 1–2, phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit (PIK3C) A–D, phosphoinositide-3-kinase regulatory subunit 1, Ras-related C3 botulinum toxin substrate 2, Raf-1 proto-oncogene serine/threonine kinase 1 and Vav guanine nucleotide exchange factor 3. PIK3R1 and Raf-1 proto-oncogene serine/threonine kinase 1 were found to be significantly upregulated in both PB- and CB-derived NK cells, whereas LCP2, PIK3CA and Ras-related C3 botulinum toxin substrate 1 were found to be significantly upregulated in only PB-derived NK cells and MAPK1 and NFATC1 genes were upregulated in only CB-derived NK cells (P < 0.05, n = 5) (see supplementary Figure 2).
      The SHC adaptor protein 2 gene associated with cytokine production, DAP12 signaling-related genes and spleen-associated tyrosine kinase and phospholipase C gamma 2 genes associated with NK cell cytotoxicity showed downregulation of expression in both donor sources (Fig. 3H). Significant downregulation of phospholipase C gamma 2 was observed in both PB- and CB-derived NK cells; however, SHC adaptor protein 2 was only significantly downregulated in CB-derived NK cells (P < 0.05, n = 5) (see supplementary Figure 2).
      The authors also observed that a few genes (KIR2DL4, KLRD1 and IFNAR2) showed an opposite trend depending on donor source. KIR2DL4 was significantly downregulated in PB-derived NK cells but showed minor upregulation in CB-derived NK cells (see supplementary Figure 2C). KLRD1 showed significant upregulation in PB-derived NK cells but showed downregulation in CB-derived NK cells. Similarly, the IFNAR2 gene was significantly downregulated in PB-derived NK cells, whereas it was upregulated (although not significantly so) in CB-derived NK cells. These results illustrated that some genes were indeed more susceptible to TGF-β exposure depending on the donor source.
      Ex vivo-expanded NK cells from CB and PB had similar decreases in proliferation, activating receptor expression and perforin secretion following TGF-β treatment. To assess the sensitivity of PB- and CB-derived NK cell functionality in response to exogenous TGF-β, treated and untreated NK cells were evaluated for their proliferation, activation marker expression and cytolytic function in response to exogenous TGF-β and the U87 MG tumor cell line, which secretes TGF-β (see supplementary Figure 4). NK cells were counted using a Luna-FL fluorescence cell counter (Logos Biosystems, Anyang, South Korea) at the beginning and end of treatment. Both CB- and PB-derived NK cell cultures had reduced cell counts in the presence of 5 ng/mL exogenous TGF-β (Fig. 4A). Specifically, PB-derived NK cells showed significantly decreased expansion (1.724–0.5457-fold, P < 0.05, n = 5) in the presence of TGF-β. Similarly, CB-derived NK cells also showed reduced expansion in the presence of TGF-β (1.395–0.5361-fold, P = 0.0642, n = 5). This effect of TGF-β on CB- versus PB-derived NK cell expansion was not significant (P > 0.05, n = 5). To test the effect of TGF-β on NK cell activating receptor expression in response to stimuli, the authors evaluated receptor expression by flow cytometry in the presence and absence of exogenous TGF-β.
      Both PB- and CB-derived NK cells showed downregulation of NK cell receptor expression (Fig. 4B,D). Specifically, for NKG2D, there was a reduction in expression from 99.71% to 69.25% on PB-derived NK cells (P < 0.05, n = 5) and from 99.51% to 57.61% on CB-derived NK cells (P < 0.05, n = 5) in the presence of TGF-β. No significant differences were found in TGF-β-mediated downregulation of NKG2D between the two donor NK cell sources (P > 0.05, n = 5) (Fig. 4B). NKp30 expression reduced from 96.27% to 46.32% on PB-derived NK cells (P < 0.05, n = 4) and from 96.57% to 52.20% on CB-derived NK cells (P < 0.05, n = 5) in the presence of TGF-β. Again, no significant differences were found between donor sources (P > 0.05, n = 5) (Fig. 4C). DNAM expression also fell from 92.59% to 37.14% on PB-derived NK cells (P < 0.05, n = 5) and from 85.98% to 26.22% on CB-derived NK cells (P < 0.05, n = 5). No significant differences were found in TGF-β-mediated downregulation of DNAM between the donor sources (P > 0.05, n = 5) (Fig. 4D). The authors subsequently assessed whether secretion of IFN-γ (Fig. 4E), granzyme (Fig. 4F) and perforin (Fig. 4G) was similarly affected.
      Cytokine secretion showed a decrease in magnitude on exposure to TGF-β. Mean IFN-γ secretion decreased from 201.8 pg/mL to 76.97 pg/mL for PB-derived NK cells and from 48.32 pg/mL to 10.72 pg/mL in CB-derived NK cells on exposure to exogenous TGF-β (n = 4). IFN-γ secretion, even though not significant, followed the same trend as mRNA expression following TGF-β exposure (Fig. 4E). Granzyme B secretion significantly decreased from 2551 pg/mL to 1568 pg/mL for PB-derived NK cells (P < 0.05, n = 4) and from 2398 pg/mL to 1719 pg/mL for CB-derived NK cells in the presence of TGF-β (n = 4). The change in granzyme B secretion (decrease) on exposure to exogenous TGF-β was more predominant than what was observed with mRNA expression (Fig. 4F). Perforin secretion decreased from 21 310 pg/mL to 17 532 pg/mL for PB-derived NK cells and from 21 913 pg/mL to 16 164 pg/mL for CB-derived NK cells (n = 4). The authors found no significant difference in cytokine expression between donor sources (P > 0.05). The decrease in perforin secretion was in concordance with the downregulation observed in perforin mRNA expression (Fig. 4G). TGF-β-treated and untreated NK cells were evaluated for their functionality by luciferase-based cytotoxicity assay against the TGF-β-secreting U87 MG cell line. Both PB- and CB-derived NK cells showed a significant decrease in cytotoxicity potential in the presence of exogenous TGF-β (P < 0.05) (Fig. 5A,B). CB-derived NK cells showed a decrease of 77.89%, and PB-derived NK cells showed a decrease of 68.53%, killing at 10:1 E:T against U87 MG cells. PB- and CB-derived NK cell products lost their killing ability at the 5:1 E:T ratio after exposure to exogenous TGF-β. However, there was no donor source difference in the TGF-β-mediated abrogation of cytolytic activity between PB- and CB-derived NK cells (P > 0.05, n = 5) (Fig. 5C).
      Ex vivo-expanded NK cells from CB and PB demonstrated similar decreases in mammalian target of rapamycin (mTOR) signaling following TGF-β treatment. Finally, evidence suggested that TGF-β-mediated inhibition of NK cell function was facilitated by repression of mTOR signaling [
      • Viel S.
      • et al.
      TGF-β inhibits the activation and functions of NK cells by repressing the mTOR pathway.
      ]. The authors therefore tested whether mTOR signaling was differentially affected in expanded CB- versus PB-derived NK cells by measuring mTOR-regulated expression of nutrient receptors CD71 [
      • Marçais A.
      • et al.
      The metabolic checkpoint kinase mTOR is essential for IL-15 signaling during the development and activation of NK cells.
      ] and CD122 [
      • Viel S.
      • et al.
      TGF-β inhibits the activation and functions of NK cells by repressing the mTOR pathway.
      ]. The authors observed comparable downregulation of CD71 in the presence of exogenous TGF-β for both PB- and CB-derived NK cells. PB-derived NK cells showed a decrease in expression from 94.79% to 70.85% (P < 0.05, n = 5), and CB-derived NK cells showed a decrease in expression from 85.30% to 48.12% (P < 0.05, n = 5) (Fig. 6A). CB-derived NK cells appeared more sensitive to TGF-β-mediated downregulation of CD71 compared with PB-derived NK cells (P < 0.05, n = 5). The authors also observed comparable downregulation in CD122 expression in the presence of exogenous TGF-β for both PB- and CB-derived NK cells. For PB-derived NK cells, CD122 expression decreased from 9.51% to 6.5% in the presence of TGF-β, and for CB-derived NK cells, expression decreased from 8.966% to 4.317% in the presence of TGF-β. However, no specific difference was found in CD122 expression between the two NK cell donor sources (P > 0.05, n = 5) (Fig. 6B).

      Discussion

      Despite promising pre-clinical results in the evaluation of NK cells as an anti-cancer therapy, their widespread clinical application has been somewhat limited as a result of several factors, including difficulties maintaining cytolytic activity in a TGF-β-rich tumor microenvironment [
      • Yvon E.S.
      • et al.
      Cord blood natural killer cells expressing a dominant negative TGF-beta receptor: implications for adoptive immunotherapy for glioblastoma.
      ,
      • Viel S.
      • et al.
      TGF-β inhibits the activation and functions of NK cells by repressing the mTOR pathway.
      ,
      • Burga R.A.
      • et al.
      Engineering the TGFβ receptor to enhance the therapeutic potential of natural killer cells as an immunotherapy for neuroblastoma.
      ,
      • Powell A.B.
      • et al.
      Medulloblastoma rendered susceptible to NK-cell attack by TGFβ neutralization.
      ]. Sham et al. have demonstrated that the cytotoxic functions of tumor-infiltrated NK cells are significantly impaired, indicating their susceptibility to the immunosuppressive tumor microenvironment [
      • Shaim H.
      • et al.
      Targeting the αv integrin/TGF-β axis improves natural killer cell function against glioblastoma stem cells.
      ]. Although the inhibitory effects of TGF-β on ex vivo-expanded NK cells have been well described [
      • Yvon E.S.
      • et al.
      Cord blood natural killer cells expressing a dominant negative TGF-beta receptor: implications for adoptive immunotherapy for glioblastoma.
      ,
      • Viel S.
      • et al.
      TGF-β inhibits the activation and functions of NK cells by repressing the mTOR pathway.
      ,
      • Burga R.A.
      • et al.
      Engineering the TGFβ receptor to enhance the therapeutic potential of natural killer cells as an immunotherapy for neuroblastoma.
      ,
      • Powell A.B.
      • et al.
      Medulloblastoma rendered susceptible to NK-cell attack by TGFβ neutralization.
      ,
      • Otegbeye F.
      • et al.
      Inhibiting TGF-beta signaling preserves the function of highly activated, in vitro expanded natural killer cells in AML and colon cancer models.
      ,
      • Shaim H.
      • et al.
      Targeting the αv integrin/TGF-β axis improves natural killer cell function against glioblastoma stem cells.
      ,
      • Wilson E.B.
      • et al.
      Human tumour immune evasion via TGF-β blocks NK cell activation but not survival allowing therapeutic restoration of anti-tumour activity.
      ], to the authors’ knowledge, the inherent differences between PB- and CB-derived NK cell responses to TGF-β have not been studied in detail. This knowledge may be a critical factor in selecting the best allogeneic cell source. A few studies have demonstrated that NK cells isolated from CB are immature and have decreased functional activity, impaired immunological synapse and increased capacity for programmed cell death compared with NK cells isolated from PB [
      • Xing D.
      • et al.
      Cord blood natural killer cells exhibit impaired lytic immunological synapse formation that is reversed with IL-2 ex vivo expansion.
      ,
      • Shereck E.
      • et al.
      Immunophenotypic, cytotoxic, proteomic and genomic characterization of human cord blood vs. peripheral blood CD56(Dim) NK cells.
      ]. This study is the first to show the comparable effects of TGF-β-mediated immune suppression between CB- and PB-derived NK cells.
      The authors’ bulk RNA sequencing of CB- and PB-derived MNCs showed significant downregulation of activating and inhibitory receptors and NK cell expression in CB-derived MNCs, which agrees with other studies [
      • Shereck E.
      • et al.
      Immunophenotypic, cytotoxic, proteomic and genomic characterization of human cord blood vs. peripheral blood CD56(Dim) NK cells.
      ]. CB-derived NK cells appear to be immature in development and have decreased translational and transcriptional genes. An increase in activity of TGF-β and the mTOR pathway further suggests that CB-derived NK cells may be more susceptible to TGF-β.
      The impaired functionality of CB-derived NK cells can be successfully reversed with ex vivo expansion in the presence of cytokines. Expanded CB-derived NK cells were comparable to PB-derived NK cells with regard to phenotype and cytolytic potential against TGF-β tumor cells (e.g., U87 MG cell line). Both PB- and CB-derived NK cells showed similar upregulation of proximal mTOR signaling receptors pSMAD2/3. Furthermore, the decrease in SMAD signaling-dependent metabolites (CD71 and CD122) in response to exogenous TGF-β was not different between donor sources. It was also evident that exposure to a TGF-β-rich microenvironment had an equivalent detrimental effect on the cytolytic activity of both PB- and CB-derived NK cell products.
      TGF-β-mediated downregulation of NK cell-activating receptors allows tumor cells to escape immune surveillance, as evidenced by the marked downregulation of the activating receptors NKG2D, DNAM1 and NKp30 on both CB- and PB-derived NK cells [
      • Crane C.A.
      • et al.
      TGF-β downregulates the activating receptor NKG2D on NK cells and CD8+ T cells in glioma patients.
      ]. The authors’ results demonstrate that there is a loss of effector function (i.e., cytotoxicity and activating receptor downregulation) after exposure to TGF-β. These data concur with earlier works published by the authors’ group and others evaluating either CB-derived [
      • Yvon E.S.
      • et al.
      Cord blood natural killer cells expressing a dominant negative TGF-beta receptor: implications for adoptive immunotherapy for glioblastoma.
      ,
      • Burga R.A.
      • et al.
      Engineering the TGFβ receptor to enhance the therapeutic potential of natural killer cells as an immunotherapy for neuroblastoma.
      • Powell A.B.
      • et al.
      Medulloblastoma rendered susceptible to NK-cell attack by TGFβ neutralization.
      ] or PB-derived [
      • Otegbeye F.
      • et al.
      Inhibiting TGF-beta signaling preserves the function of highly activated, in vitro expanded natural killer cells in AML and colon cancer models.
      ,
      • Wilson E.B.
      • et al.
      Human tumour immune evasion via TGF-β blocks NK cell activation but not survival allowing therapeutic restoration of anti-tumour activity.
      ,
      • Zhao Y.
      • et al.
      Enhanced NK cell adoptive antitumor effects against breast cancer in vitro via blockade of the transforming growth factor-β signaling pathway.
      ] NK cells.
      The unbiased clustering of individual gene expression changes in the presence of exogenous TGF-β revealed that ex vivo-expanded NK cells showed only modest clustering according to donor source, further confirming that ex vivo expansion potentially neutralizes the inherent differences between CB- and PB-derived NK cells. Additionally, the authors observed an upregulation of inhibitory receptors (e.g., growth factor receptor-bound protein 2, KIR3DL1, KLRC1 and protein tyrosine phosphatase non-receptor type 6), revealing a novel mechanism of TGF-β-mediated immune suppression not previously reported. As expected, the authors observed the downregulation of many essential cytotoxicity pathways in expanded NK cells, including the lytic enzyme perforin as well as NCR2, NCR3 and integrin subunit beta 2, irrespective of donor source. The authors further observed that some essential pathways associated with NK cell cytotoxicity, including granzyme B, IFNAR1, 2B4 and FCE31G, were still upregulated with exposure to exogenous TGF-β, indicating that NK cells were not completely functionally impaired post-exposure to TGF-β. This could explain the residual cytotoxicity observed after TGF-β exposure. The overall upregulation trend of NK cell effector function pathways (specifically, LCK proto-oncogene Src family tyrosine kinase, LCP2, MAPK1 and NFATC1) further confirmed this assertion. Together, these results further suggest that TGF-β-mediated immune suppression does not render NK cells completely dysfunctional.
      It is important to point out that the authors observed some discrepancies in the consistency of upregulation/downregulation between both NK cell donor sources. Some pathways were significantly upregulated (e.g., KIR2DS3, KIR3DL1, TNFSF10B and LCP2) or downregulated (e.g., perforin, KLRD1 and KIR2DL4) in one source but not in the other. The authors also observed a switch between an upregulation and downregulation trend between the two donor sources. For example, KIR2DL4 associated with NK cell's inhibitory pathway gene was upregulated in CB-derived NK cells, whereas it showed significant downregulation in PB-derived NK cells. Similarly, KLRD1 was significantly upregulated in PB-derived NK cells but showed downregulation in CB-derived NK cells. Further, IFNAR2, associated with cytokine pathways, showed significant downregulation in PB-derived NK cells but was upregulated in CB-derived NK cells. These data support the development and translation of strategies to restore NK cell function in the presence of TGF-β, including the use of TGF-β-neutralizing antibodies or a TGF-β signaling inhibitor like LY2157299 (TGF-β kinase inhibitor) [
      • Otegbeye F.
      • et al.
      Inhibiting TGF-beta signaling preserves the function of highly activated, in vitro expanded natural killer cells in AML and colon cancer models.
      ] or NK cell gene engineering strategies using a dominant negative TGF-β receptor to mitigate TGF-β signaling [
      • Yvon E.S.
      • et al.
      Cord blood natural killer cells expressing a dominant negative TGF-beta receptor: implications for adoptive immunotherapy for glioblastoma.
      ,
      • Powell A.B.
      • et al.
      Medulloblastoma rendered susceptible to NK-cell attack by TGFβ neutralization.
      ,
      • Bollard C.M.
      • et al.
      Adapting a transforming growth factor β–related tumor protection strategy to enhance antitumor immunity.
      ] or deleting TGFBR2 on NK cells [
      • Shaim H.
      • et al.
      Targeting the αv integrin/TGF-β axis improves natural killer cell function against glioblastoma stem cells.
      ].

      Conclusions

      In summary, these results indicate that some pathways may be more susceptible/sensitive to TGF-β exposure in one donor source over the other, underlining some mechanistic differences between PB- and CB-derived NK cells. In addition to these intrinsic differences, the authors also observed a donor-dependent discrepancy in gene expression change on exposure to TGF-β. This indicates that some donors, irrespective of the donor source, are more resistant or sensitive to TGF-β-mediated immune suppression. Further exploration of such “sensitive” donors could inform essential screening or release criteria to prepare more resistant NK cell products for cancer immunotherapy. Overall, the relative change in gene expression in both CB- and PB-derived NK cells was equivalent, which further confirms the authors’ hypothesis that both donor sources are equally susceptible to TGF-β-mediated immune suppression.

      Funding

      This work was partially supported by Catamaran Bio (LLC), National Cancer Institute (NCI) grant (P30CA016056) involving the use of Roswell Park Comprehensive Cancer Center's Genomic Shared Resource, and the NIH Moonshot grant (5 U01 CA239258-02)

      Author contributions

      Conception and design of the study: KC, CMB, CRYC, PS, SL and MDL. Acquisition of data: KC, ED, CS, AG, HL, NM and PS. Analysis and interpretation of data: KC, CMB, CRYC, PS, SL and MD. Drafting or revising the manuscript: KC, CMB and CRYC. All authors have approved the final article.

      Declaration of Competing Interest

      CRYC is a consultant for the NK cell company Catamaran Bio. CMB is a co-founder and scientific advisory board member for Catamaran Bio. CRYC, CS and CMB have intellectual property related to the development of NK cell therapies.

      Gene annotation

      Tabled 1
      Gene ID usedGene Name
      ITGB2integrin subunit beta 2(ITGB2)
      PRF1perforin 1(PRF1)
      PIK3CDphosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit delta(PIK3CD)
      FASLGFas ligand(FASLG)
      PIK3CBphosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit beta(PIK3CB)
      ITGALintegrin subunit alpha L(ITGAL)
      TNFtumor necrosis factor(TNF)
      ACTBactin beta(ACTB)
      FCGR3AFc fragment of IgG receptor IIIa(FCGR3A)
      FCGR3BFc fragment of IgG receptor IIIb(FCGR3B)
      TNFSF10tumor necrosis factor superfamily member 10(TNFSF10)
      RAC2ras-related C3 botulinum toxin substrate 2 (rho family, small GTP binding protein Rac2)(RAC2)
      RAC3ras-related C3 botulinum toxin substrate 3 (rho family, small GTP binding protein Rac3)(RAC3)
      RAC1ras-related C3 botulinum toxin substrate 1 (rho family, small GTP binding protein Rac1)(RAC1)
      GUSBglucuronidase beta(GUSB)
      B2Mbeta-2-microglobulin(B2M)
      IFNAR2interferon alpha and beta receptor subunit 2(IFNAR2)
      VAV3vav guanine nucleotide exchange factor 3(VAV3)
      FCER1GFc fragment of IgE receptor Ig(FCER1G)
      SYKspleen associated tyrosine kinase(SYK)
      RPL13Aribosomal protein L13a(RPL13A)
      VAV1vav guanine nucleotide exchange factor 1(VAV1)
      VAV2vav guanine nucleotide exchange factor 2(VAV2)
      NCR1natural cytotoxicity triggering receptor 1(NCR1)
      ZAP70zeta chain of T cell receptor associated protein kinase 70(ZAP70)
      NCR2natural cytotoxicity triggering receptor 2(NCR2)
      NCR3natural cytotoxicity triggering receptor 3(NCR3)
      TYROBPTYRO protein tyrosine kinase binding protein(TYROBP)
      IFNGinterferon gamma(IFNG)
      PIK3CAphosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha(PIK3CA)
      LCKLCK proto-oncogene, Src family tyrosine kinase(LCK)
      LCP2lymphocyte cytosolic protein 2(LCP2)
      HPRT1hypoxanthine phosphoribosyltransferase 1(HPRT1)
      RAF1Raf-1 proto-oncogene, serine/threonine kinase(RAF1)
      PPIApeptidylprolyl isomerase A(PPIA)
      GAPDHglyceraldehyde-3-phosphate dehydrogenase(GAPDH)
      IFNAR1interferon alpha and beta receptor subunit 1(IFNAR1)
      SHC2SHC adaptor protein 2(SHC2)
      SHC1SHC adaptor protein 1(SHC1)
      PIK3R3phosphoinositide-3-kinase regulatory subunit 3(PIK3R3)
      PIK3R2phosphoinositide-3-kinase regulatory subunit 2(PIK3R2)
      KIR2DL1killer cell immunoglobulin like receptor, two Ig domains and long cytoplasmic tail 1(KIR2DL1)
      PIK3R1phosphoinositide-3-kinase regulatory subunit 1(PIK3R1)
      KIR2DL2killer cell immunoglobulin like receptor, two Ig domains and long cytoplasmic tail 2(KIR2DL2)
      KIR2DL3killer cell immunoglobulin like receptor, two Ig domains and long cytoplasmic tail 3(KIR2DL3)
      KIR2DL4killer cell immunoglobulin like receptor, two Ig domains and long cytoplasmic tail 4(KIR2DL4)
      PAK1p21 (RAC1) activated kinase 1(PAK1)
      SH3BP2SH3 domain binding protein 2(SH3BP2)
      PLCG2phospholipase C gamma 2(PLCG2)
      PGK1phosphoglycerate kinase 1(PGK1)
      MAPK1mitogen-activated protein kinase 1(MAPK1)
      FYNFYN proto-oncogene, Src family tyrosine kinase(FYN)
      PLCG1phospholipase C gamma 1(PLCG1)
      KLRC1killer cell lectin like receptor C1(KLRC1)
      MAPK3mitogen-activated protein kinase 3(MAPK3)
      KIR2DL5Akiller cell immunoglobulin like receptor, two Ig domains and long cytoplasmic tail 5A(KIR2DL5A)
      KIR2DS1killer cell immunoglobulin like receptor, two Ig domains and short cytoplasmic tail 1(KIR2DS1)
      KIR2DS2killer cell immunoglobulin like receptor, two Ig domains and short cytoplasmic tail 2(KIR2DS2)
      KIR2DS3killer cell immunoglobulin like receptor, two Ig domains and short cytoplasmic tail 3(KIR2DS3)
      SH2D1ASH2 domain containing 1A(SH2D1A)
      KIR2DS4killer cell immunoglobulin like receptor, two Ig domains and short cytoplasmic tail 4(KIR2DS4)
      KIR2DS5killer cell immunoglobulin like receptor, two Ig domains and short cytoplasmic tail 5(KIR2DS5)
      GZMBgranzyme B(GZMB)
      TNFRSF10BTNF receptor superfamily member 10b(TNFRSF10B)
      NFATC2nuclear factor of activated T-cells 2(NFATC2)
      PTPN11protein tyrosine phosphatase, non-receptor type 11(PTPN11)
      KIR3DL1killer cell immunoglobulin like receptor, three Ig domains and long cytoplasmic tail 1(KIR3DL1)
      NFATC1nuclear factor of activated T-cells 1(NFATC1)
      KIR3DL2killer cell immunoglobulin like receptor, three Ig domains and long cytoplasmic tail 2(KIR3DL2)
      FASFas cell surface death receptor(FAS)
      GRB2growth factor receptor bound protein 2(GRB2)
      PTPN6protein tyrosine phosphatase, non-receptor type 6(PTPN6)
      KLRD1killer cell lectin like receptor D1(KLRD1)
      LATlinker for activation of T-cells(LAT)
      2B4Natural Killer Cell Receptor 2B4

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