ER-Stress-Induced Suppression of HLA-E on Bortezomib-Evading Malignant Plasma Cells Dramatically Enhances Their Susceptibility to NK Cell Killing: Identification of an Achilles Heel in Myeloma Cells That Can be Utilized to Prevent Disease Relapse Following Bortezomib Treatment

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 4296-4296
Author(s):  
Mattias Carlsten ◽  
Ali Namazi ◽  
Robert N. Reger ◽  
Maria Berg ◽  
Richard W. Childs
Keyword(s):  
Er Stress ◽  
Nk Cells ◽  
Nk Cell ◽  
Ex Vivo ◽  
Hla Class I ◽  
Proteasome Inhibition ◽  
Cell Killing ◽  
Class I ◽  
Term Survival ◽  
Myeloma Cells

Abstract Although the proteasome inhibitor bortezomib has significantly improved the survival of patients with multiple myeloma (MM), most patients treated with this drug eventually develop progressive disease. Recent data indicate that MM cells can evade bortezomib-induced cell death by undergoing autophagy as a consequence of endoplasmatic reticulum (ER)-stress triggered by proteasome inhibition. Here we show that bortezomib-evading MM cells become highly sensitized to killing by natural killer (NK) cells via ER-stress-induced reduction of the NK cell inhibitory molecule HLA-E that is normally expressed at high levels on the surface of MM cells. High-resolution flow cytometry-based assays revealed augmented NK cell recognition and degranulation against bortezomib-exposed MM cells (3 fold higher compared to untreated MM controls) was restricted to NK cells exclusively controlled by the HLA-E-binding inhibitory receptor NKG2A (NKG2ASP NK cells) (Figure 1). In contrast, due to unchanged high expression of other HLA class I molecules on the surface of bortezomib-exposed MM cells there was no augmentation in degranulation by NK cells controlled by other inhibitory HLA class I-binding receptors, such as killer immunoglobulin-like receptors (KIRs). Compared to their non-expanded counterparts, ex vivo expanded NK cells have previously been shown to have an increased proportion of NKG2ASP NK cells (50% vs 25%, p<0.01) and bolstered cytotoxic function against tumor cells. Using NK cells expanded in accordance with our ongoing FDA-approved clinical trial evaluating adoptive infusion of autologous ex vivo expanded NK cells in patients with refractory cancers at the NIH, we established these highly cytotoxic NKG2ASP dominant NK cells induced substantially higher lysis of bortezomib-exposed MM cells compared to non-expanded matched control NK cells (38% vs 18%, p<0.05) (Figure 2). Based on these findings, we hypothesize that adoptive infusions of ex vivo expanded autologous NK cells following treatment with bortezomib could eradicate the fraction of MM cells that would normally evade killing through proteasome inhibition alone. The increased vulnerability of bortezomib-evading cells to NK cell cytotoxicity identifies a critical Achilles heel in myeloma cells and suggests adoptive NK cell infusions following bortezomib therapy could potentially be utilized as a strategy to improve long-term survival in MM patients. Figure 1. Degranulation of NK cell subsets against myeloma cells exposed to bortezomib. NT; No target NK cell killing of bortezomib-exposed myeloma cells. Figure 1. Degranulation of NK cell subsets against myeloma cells exposed to bortezomib. NT; No target. / NK cell killing of bortezomib-exposed myeloma cells. Disclosures No relevant conflicts of interest to declare.

Treatment of Ex Vivo Expanded NK Cells with Daratumumab F(ab')2 Fragments Protects Adoptively Transferred NK Cells from Daratumumab-Mediated Killing and Augments Daratumumab-Induced Antibody Dependent Cellular Toxicity (ADCC) of Myeloma

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 4244-4244 ◽  
Author(s):  
Elena Cherkasova ◽  
Luis Espinoza ◽  
Ritesh Kotecha ◽  
Robert N. Reger ◽  
Maria Berg ◽  
...  
Keyword(s):  
Nk Cells ◽  
Adoptive Transfer ◽  
Nk Cell ◽  
Ex Vivo ◽  
Cell Killing ◽  
Cellular Toxicity ◽  
Myeloma Cells ◽  
Nsg Mice

Abstract Daratumumab is a fully humanized monoclonal antibody (IgG1) that targets CD38 expressed on myeloma cells. Daratumumab kills myeloma cells through antibody dependent cellular toxicity (ADCC), compliment dependent cytotoxicity (CDC), and antibody dependent phagocytosis (ADCP). In early clinical trials, daratumumab has showed significant anti-myeloma activity in patients with treatment refractory disease. In vivo, daratumumab has been found to induce NK cell lymphopenia of unclear etiology. We found that NK cells isolated from the peripheral blood of healthy and cancer patients expressed variable surface levels of CD38 (Fig. 1A). Further, surface expression of CD38 increased substantially when NK cells underwent ex vivo cytokine activation by culturing cells overnight in IL-2 containing media or ex vivo expansion using irradiated EBV-LCL feeder cells (Fig. 1B). Remarkably, daratumumab induced apoptosis of expanded NK cells in a dose dependent manner, with substantial NK cell apoptosis occurring within 2 hours following in vitro exposure to daratumumab at a concentration of 1 and 10 ug/ml (Fig. 1C). Further, adoptive transfer of ex vivo expanded human NK cells into NSG mice that had been pre-treated with daratumumab showed daratumumab induced NK cell killing in vivo: the numbers of NK cells isolated from the lungs, blood, spleen and bone marrow of NSG mice 24 hours after infusion of expanded human NK cells was reduced by 90% in mice that were pretreated with 1 mg/kg of daratumumab i.p. compared to controls that had not received the antibody (Fig. 1D). In vitro experiments showed NK cell killing by daratumumab occurred as a consequence of ADCC and was dependent on NK cell CD16 expression; when CD56+ NK cells were sorted by FACS into CD16 positive and negative populations, only NK cells expressing CD16 were killed by daratumumab, with no effect on NK cell viability occurring in the CD16- NK cell. Further, we observed that NK cells obtained from donors who have high affinity FCgR3 as a consequence of a single nucleotide polymorphism in the FCGR3A gene resulting in an amino acid substitution at position 158 (F158V) in CD16 were more sensitive to daratumumab killing compared to NK cells isolated from donors carrying the low affinity CD16 polymorphism. Although NK cell counts and NK reduction in peripheral blood and bone marrow were not associated with daratumumab clinical response in myeloma studies, NK cells play an important role in mediating antitumor responses through ADCC following mAb therapy. In this regard, combining mAb therapy with adoptive transfer of ex vivo expanded NK cells could be utilized as a strategy to potentiate the antitumor effects of mAbs. To overcome daratumumab-mediated killing of adoptively transferred NK cells in daratumumab-treated patients, we blocked CD38 on the surface of NK cells by pretreating them with daratumumab F(ab')2 fragments. The F(ab')2 fragments that were generated using pepsin cleavage of daratumumab were confirmed to bind and block the CD38 epitope expressed on NK cells. Importantly, these F(ab')2 fragments remained bound to the surface of NK cells for at least 96 hours, did not induce NK cell apoptosis, protected NK cells from daratumumab-mediated NK cell killing, and bolstered their tumor cytotoxicity against daratumumab-treated myeloma targets. In vitro experiments showed NK cell tumor cytotoxicity vs myeloma cells in daratumumab-containing media was significantly higher by NK cells that had CD38 blocked with F(ab')2 fragments compared to unblocked controls (Fig. 1E). Importantly, pretreatment with daratumumab F(ab')2 fragments also protected human NK cells from daratumumab-mediated killing in vivo; expanded NK cells pretreated with F(ab')2 fragments prior to adoptive transfer into NSG mice that had been treated with daratumumab were detectable at significantly higher numbers in the blood compared to untreated NK cell controls (Fig. 1F). Conclusion: Expression of CD38 on activated NK cells makes them susceptible to killing by daratumumab, which could compromise the ability of adoptively transferred NK cells to bolster ADCC following treatment with this mAb. Pretreatment of ex vivo expanded NK cells with daratumumab F(ab')2 fragments protects cells from daratumumab-mediated killing, potentially offering a strategy to augment the anti-tumor effects of adoptively transferred NK cells in myeloma patients that have received daratumumab treatment. Disclosures No relevant conflicts of interest to declare.

scholarly journals Ex Vivo Expanded NK Cells Mediate Highly Efficient and Rapid Killing of Ewing Sarcoma Cells Through Degranulation with Tumor Cytotoxicity Controlled by the NKG2D, DNAM-1, and NKp30 NK Receptors

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 1894-1894
Author(s):  
Amrita D. Karambelkar ◽  
Robert N. Reger ◽  
Mattias Carlsten ◽  
Richard W. Childs
Keyword(s):  
Nk Cells ◽  
Gold Standard ◽  
Nk Cell ◽  
Ex Vivo ◽  
Hla Class I ◽  
K562 Cells ◽  
Class I ◽  
Nk Cell Receptors ◽  
Hla Class I Molecules ◽  
Cell Target

Abstract Introduction : Natural killer (NK) cells are highly cytotoxic immune cells that can kill tumor cells via release of cytotoxic granulae as well as through induction of tumor apoptosis by ligands that bind death receptors expressed on the target cells. Clinical trials have established that adoptive infusions of ex vivo expanded NK cells are safe and can induce tumor regression in selected groups of cancer patients. Recent data suggest that Ewing's sarcoma (EwS), a bone cancer associated with poor survival in the context of metastatic disease, is exquisitely sensitive to killing by NK cells due to low expression of HLA class I molecules that normally prevent NK cell cytotoxicity through interactions with inhibitory NK cell receptors. We and others have recently shown that ex vivo expansion of NK cells causes upregulation of their activation receptors such as NKG2D and death receptor ligands such as TRAIL, which collectively make expanded NK cells more cytotoxic than resting non-expanded NK cells. In an effort to optimize the full therapeutic potential of adoptive NK cell immunotherapy against EwS in the clinic, we investigated the mechanisms utilized by ex vivo expanded NK cells to recognize and kill EwS cells. Methods : Healthy donorNK cells were expanded for 14 days using irradiated EBV-LCL cells in X-Vivo 20 media supplemented with 500 IU/ml IL-2 and 10% AB serum. The EwS cell lines (TC71, RH18X, LG) and the K562 cell line were grown in RPMI media supplemented with 10% FBS. NK cell viability, phenotype, and degranulation were measured by flow cytometry. EwS lysis was measured using 51 Cr release assays. Degradation of perforin to prevent tumor killing via the degranulation pathway was achieved by pre-treating NK cells for 2 hours with 100 nM concanamycin. Blocking antibodies against HLA-A,B,C antigens on EwS cells and against activation receptors on NK cells were added to the respective cells for 30-45 min prior to co-culture. In some experiments, EwS cells were pre-treated with 20 nM bortezomib for 24 hours prior to co-culture with NK cells. Statistical analysis was conducted using the Wilcoxon ranked sum test to determine significance. Results: Ex vivo expanded NK cells were highly cytotoxic against all three EwS cell lines tested, with killing levels comparable to those of the gold-standard NK cell target K562 cells. Suppression of the degranulation pathway using concanamycin revealed a significant reduction in the ability of NK cells to lyse EwS cells (65-71% at baseline vs 10-24% with concanamycin-treated NK cells). Blockade of HLA class I molecules on the EwS cell surface revealed a small but significant increase in NK cell degranulation from 30 to 37%, 32 to 40%, and 20 to 35% against the TC71, RH18X, and LG EwS lines respectively (p <0.05). Based on experiments where individual activation receptors on ex vivo expanded NK cells were blocked with antibodies, we established that EwS killing by these cells was highly dependent on the expression of the NKG2D, DNAM-1, and NKp30 receptors. Although blockade of individual receptors significantly reduced NK cell killing of EwS cells, simultaneous blockade of all three receptors completely prevented NK cell degranulation. In an attempt to further bolster NK cell killing of EwS cells, we next pre-treated EwS cells with the proteasome inhibitor bortezomib to increase the expression of the TRAIL receptor DR5. While this approach increased DR5 expression by a median 2.09 fold (range 1.40-2.15) and enhanced the susceptibility of EwS cells to killing by recombinant TRAIL, surprisingly, no further killing was observed following co-culture with expanded NK cells. Preliminary data indicate the latter is explained by the rapid and efficient EwS killing induced by NK cell degranulation that triggers instant lysis in contrast to more delayed killing that is characteristic of the TRAIL pathways. Conclusions: Ex vivo expanded NK cells are able to rapidly and efficiently kill EwS cells at levels comparable to those of the gold-standard NK cell target K562 cells. Lysis of EwS by ex vivo expanded NK cells occurs exclusively through degranulation triggered by a relative lack of HLA class I expression combined with expression of ligands to the activating NK cell receptors NKG2D, DNAM-1, and NKp30. These data provide important insights that define the critical elements required by ex vivo expanded NK cells to mediate tumor responses against metastatic EwS following adoptive transfer in the clinic. Disclosures No relevant conflicts of interest to declare.

Anti-KIR (1-7F9): A Fully Human Monoclonal Antibody (mAb) That Blocks KIR2DL1, −2 and −3, Promoting Natural Killer (NK) Cell-Mediated Lysis of Tumor Cells In Vitro and In Vivo.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 582-582 ◽  
Author(s):  
Nicolai Wagtmann ◽  
Pascale Andre ◽  
Stefan Zahn ◽  
Pieter Spee ◽  
Nicolas Anfossi ◽  
...  
Keyword(s):  
Nk Cells ◽  
Mononuclear Cells ◽  
Nk Cell ◽  
Ex Vivo ◽  
Phase 1 ◽  
Human Monoclonal Antibody ◽  
Hla Class I ◽  
Term Survival

Abstract In patients with acute myeloid leukemia (AML), haplo-identical stem cell transplantation (SCT) can lead to expansion and activation of Killer Immunoglobulin-like Receptor (KIR)-HLA class I mismatched NK cells, resulting in reduced rates of leukemia relapse and no graft-versus-host disease (Ruggeri et al. Science 2002). However, this SCT is not available to the majority of AML patients who are elderly. To explore the feasibility of achieving similar NK-mediated anti-leukemia activity by a pharmacological approach, we generated fully human anti-KIR mAbs that block the interactions of inhibitory KIR2DL receptors with their HLA-C ligands, thereby enhancing NK activity. Here we describe one such therapeutic candidate anti-KIR mAb, designated 1-7F9. As distinct HLA-C allotypes are recognized by KIR2DL1 or −2/3, only mAbs that cross-react with these KIRs would be expected to work in the entire population. Hence, 1-7F9 was initially selected based on its ability to bind soluble, recombinant KIR2L1, −2 and −3. By Biacore analysis, the bivalent affinities for KIR2DL1 and −3 were 0.43 × 10−9 M and 0.025 × 10−9 M, respectively. In experimental systems and in normal human blood, 1-7F9 bound KIR2DL1, −2 and −3, and −2DS1 and −2, but not to KIR2DS3 or −4. 1-7F9 dose-dependently inhibited the binding of soluble KIR2DL1-Fc to cell surface HLA-Cw4. 1-7F9 augmented the lysis of 721.221-Cw4 B-EBV cells by an NK cell line transfected with KIR2DL1 (YTS-2DL1) from 5% lysis in absence of mAb to a maximal 55% lysis at 5 ug/ml of mAb, but did not affect lysis by KIR-negative NK cells. Lysis of PHA-stimulated blasts and primary AML blasts by autologous IL-2 activated NK cells (E:T=6:1) was 10 and 15%, respectively, in absence of mAb vs 80% and 55% in presence of 1-7F9. Incubation of IL-2 activated blood mononuclear cells with 1-7F9 resulted in expression of the activation marker CD107 on about 10% of KIR2D-positive NK cells, which increased to 20% upon addition of HLA-C-positive B-EBV targets, suggesting that 1-7F9 preferentially induces activation of NK cells in presence of transformed cells. The isotype of 1-7F9 is IgG4; accordingly, it did not cause depletion of KIR positive cells in vitro or in vivo in KIR-transgenic mice despite long-lived KIR-occupancy. As KIR are not found in mice, in vivo activity was tested in a NOD-SCID mouse model where inoculation of in vitro-expanded NK cells (80% of NK cells KIR2D-positive) and autologous human B-EBV cells (E:T=1:3) resulted in death of all mice by day 26. A single injection of 1-7F9 (125 ug/mouse) resulted in long-term survival, with 100% of treated mice alive beyond day 60; in contrast, 60 ug/mouse of the mAb was ineffective. Similarly, ex vivo pre-incubation of NK cells with 1-7F9 (37,3 ug/106 NK cells) prior to inoculation in mice resulted in elimination of the autologous transformed B cells in vivo and survival of 100% of the treated animals. These data show that 1-7F9 augments NK-mediated tumor killing in vitro and in vivo, and that it exhibits long-lived KIR binding in vivo, providing a preclinical basis for initiating phase 1 clinical trials with the mAb.

Identifying NK-Cell Donors for Cell Therapy Based on Functional Phenotype.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 3271-3271
Author(s):  
Lenka Hurton ◽  
R. Iram Siddik ◽  
Harjeet Singh ◽  
Simon Olivares ◽  
Brian Rabinovich ◽  
...  
Keyword(s):  
Nk Cells ◽  
Target Cell ◽  
Nk Cell ◽  
Hla Class I ◽  
Cell Lysis ◽  
Cell Killing ◽  
Hla Typing ◽  
Class I ◽  
Target Cells ◽  
Functional Assay

Abstract Donor natural killer (NK) cells after haploidentical hematopoietic stem-cell transplantation (HSCT) and infusion of haploidentical NK-cells have demonstrated a therapeutic effect. NK alloreactivity resulting from appropriate Killer cell Ig-like receptor (KIR)-ligand disparity in human-leukocyte-antigen (HLA)-haplotype mismatched HSCT has resulted in improved engraftment and decreased incidence of leukemia relapse. Yet, not all patient-donor pairs benefit for an allogeneic NK-cell effect. To identify NK-cell donors with a suitable KIR-ligand mismatch, we have developed a functional assay to measure NK-cell killing through KIR-ligand interactions. NK-cell lysis of target cells is blocked by inhibitory KIR that recognize classical HLA class I allotypes and HLA mismatches of an altered allelic repertoire, as in haploidentical HSCT, leading to KIR-ligand mismatch and alloreactive NK cell-mediated target killing (Figure 1A). A cytotoxicity assay was developed based on the NK-cell target HLAnull 721.221 cells, and a panel of targets with enforced expression of HLA genes recognized by KIR. After the killing assay was optimized for high throughput and sensitivity, we used the panel of targets to determine whether bulk populations of donor NK cells could be predicted to kill based on KIR and HLA typing. The results demonstrate patterns of target-cell lysis for the KIR repertoires corresponding, for some donors, with predicted donor-versus-recipient NK-cell alloreactivity (Figure 1B). A relative inhibition of HLA+ target-cell lysis of >30% was associated with binding of KIR to introduced HLA class I molecules. The benefit of this assay to transplant physicians is a tool to actually measure phenotype (lysis), rather than relying on predictive models based on genotype. This assay will be combined with typing data to help identify donors with NK-cell killing function for recipients of haploidentical HSCT and infusion of haploidentical NK cells. Figure 1. (A) Schematic of alloreactivity generated between NK cells that are KIR-ligand mismatched with targets. (B) Observed lysis of 721.221 cells, with enforced expression of HLA class I, by KIR-typed donar(box). Figure 1. (A) Schematic of alloreactivity generated between NK cells that are KIR-ligand mismatched with targets. (B) Observed lysis of 721.221 cells, with enforced expression of HLA class I, by KIR-typed donar(box).

scholarly journals High-Risk Leukemia: Past, Present, and Future Role of NK Cells

2018 ◽  
Vol 2018 ◽  
pp. 1-12 ◽  
Author(s):  
Melissa Mavers ◽  
Alice Bertaina
Keyword(s):  
Acute Leukemia ◽  
Nk Cells ◽  
Nk Cell ◽  
Ex Vivo ◽  
Selection Criterion ◽  
Hla Class I ◽  
Hematopoietic Stem ◽  
Genotypic Analysis ◽  
Antileukemic Effect ◽  
Innate Lymphocytes

Natural killer (NK) cells are a population of cytotoxic innate lymphocytes that evolved prior to their adaptive counterparts and constitute one of the first lines of defense against infected/mutated cells. Several studies have shown that in patients with acute leukemia given haploidentical hematopoietic stem cell transplantation, donor-derived NK cells play a key role in the eradication of cancer cells. The antileukemic effect is mostly related to the presence of “alloreactive” NK cells, that is, mature KIR+ NK cells that express inhibitory KIR mismatched with HLA class I (KIR-L) of the patient. A genotypic analysis detecting KIR B haplotype and the relative B content is an additional donor selection criterion. These data provided the rationale for implementing phase I/II clinical trials of adoptive infusion of either selected or ex vivo-activated NK cells, often from an HLA-mismatched donor. In this review, we provide a historical perspective on the role played by NK cells in patients with acute leukemia, focusing also on the various approaches to adoptive NK cell therapy and the unresolved issues therein. In addition, we outline new methods to enhance NK activity, including anti-KIR monoclonal antibody, bi-/trispecific antibodies linking NK cells to cytokines and/or target antigens, and CAR-engineered NK cells.

scholarly journals Preclinical characterization of 1-7F9, a novel human anti–KIR receptor therapeutic antibody that augments natural killer–mediated killing of tumor cells

Blood ◽  
2009 ◽  
Vol 114 (13) ◽  
pp. 2667-2677 ◽  
Author(s):  
Francois Romagné ◽  
Pascale André ◽  
Pieter Spee ◽  
Stefan Zahn ◽  
Nicolas Anfossi ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Natural Killer ◽  
Tumor Cells ◽  
Nk Cell ◽  
Hla Class I ◽  
Therapeutic Antibody ◽  
Class I ◽  
Therapeutic Window ◽  
Target Cells ◽  
Term Survival

Abstract Inhibitory-cell killer immunoglobulin-like receptors (KIR) negatively regulate natural killer (NK) cell–mediated killing of HLA class I–expressing tumors. Lack of KIR-HLA class I interactions has been associated with potent NK-mediated antitumor efficacy and increased survival in acute myeloid leukemia (AML) patients upon haploidentical stem cell transplantation from KIR-mismatched donors. To exploit this pathway pharmacologically, we generated a fully human monoclonal antibody, 1-7F9, which cross-reacts with KIR2DL1, -2, and -3 receptors, and prevents their inhibitory signaling. The 1-7F9 monoclonal antibody augmented NK cell–mediated lysis of HLA-C–expressing tumor cells, including autologous AML blasts, but did not induce killing of normal peripheral blood mononuclear cells, suggesting a therapeutic window for preferential enhancement of NK-cell cytotoxicity against malignant target cells. Administration of 1-7F9 to KIR2DL3-transgenic mice resulted in dose-dependent rejection of HLA-Cw3–positive target cells. In an immunodeficient mouse model in which inoculation of human NK cells alone was unable to protect against lethal, autologous AML, preadministration of 1-7F9 resulted in long-term survival. These data show that 1-7F9 confers specific, stable blockade of KIR, boosting NK-mediated killing of HLA-matched AML blasts in vitro and in vivo, providing a preclinical basis for initiating phase 1 clinical trials with this candidate therapeutic antibody.

scholarly journals Specificity of HLA class I antigen recognition by human NK clones: evidence for clonal heterogeneity, protection by self and non-self alleles, and influence of the target cell type.

1993 ◽  
Vol 178 (4) ◽  
pp. 1321-1336 ◽  
Author(s):  
V Litwin ◽  
J Gumperz ◽  
P Parham ◽  
J H Phillips ◽  
L L Lanier
Keyword(s):  
Nk Cells ◽  
Mhc Class I ◽  
Target Cell ◽  
Nk Cell ◽  
Hla Class I ◽  
Class I ◽  
Target Cells ◽  
Clonal Heterogeneity ◽  
Multiple Alleles ◽  
Wide Range

Prior studies using polyclonal populations of natural killer (NK) cells have revealed that expression of certain major histocompatibility complex (MHC) class I molecules on the membrane of normal and transformed hematopoietic target cells can prevent NK cell-mediated cytotoxicity. However, the extent of clonal heterogeneity within the NK cell population and the effect of self versus non-self MHC alleles has not been clearly established. In the present study, we have generated more than 200 independently derived human NK cell clones from four individuals of known human histocompatibility leukocyte antigens (HLA) type. NK clones were analyzed for cytolytic activity against MHC class I-deficient Epstein Barr virus (EBV) transformed B lymphoblastoid cell lines (B-LCL) stably transfected with several HLA-A, -B, or -C genes representing either self or non-self alleles. All NK clones killed the prototypic HLA-negative erythroleukemia K562 and most lysed the MHC class I-deficient C1R and 721.221 B-LCL. Analysis of the panel of HLA-A, -B, and -C transfectants supported the following general conclusions. (a) Whereas recent studies have suggested that HLA-C antigens may be preferentially recognized by NK cells, our findings indicate that 70% or more of all NK clones are able to recognize certain HLA-B alleles and many also recognize HLA-A alleles. Moreover, a single NK clone has the potential to recognize multiple alleles of HLA-A, HLA-B, and HLA-C antigens. Thus, HLA-C is not unique in conferring protection against NK lysis. (b) No simple patterns of HLA specificity emerged. Examination of a large number of NK clones from a single donor revealed overlapping, yet distinct, patterns of reactivity when a sufficiently broad panel of HLA transfectants was examined. (c) Both autologous and allogeneic HLA antigens were recognized by NK clones. There was neither evidence for deletion of NK clones reactive with self alleles nor any indication for an increased frequency of NK clones recognizing self alleles. (d) With only a few exceptions, protection conferred by transfection of HLA alleles into B-LCL was usually not absolute. Rather a continuum from essentially no protection for certain alleles (HLA-A*0201) to very striking protection for other alleles (HLA-B*5801), with a wide range of intermediate effects, was observed. (e) Whereas most NK clones retained a relatively stable HLA specificity, some NK clones demonstrated variable and heterogeneous activity over time. (f) NK cell recognition and specificity cannot be explained entirely by the presence or absence of HLA class I antigens on the target cell.(ABSTRACT TRUNCATED AT 400 WORDS)

Anti-Leukemia Activity of Alloreactive NK Cells in Haploidentical HSCT in Pediatric Patients: Re-Defining the Role of Activating and Inhibitory KIR

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 3002-3002 ◽  
Author(s):  
Daniela Pende ◽  
Stefania Marcenaro ◽  
Michela Falco ◽  
Stefania Martini ◽  
Maria Ester Bernardo ◽  
...  
Keyword(s):  
T Cell ◽  
Nk Cells ◽  
Nk Cell ◽  
Cell Subset ◽  
Hla Class I ◽  
Class I ◽  
Leukemia Cells ◽  
Hla Alleles ◽  
Time Points ◽  
Selection Of

Abstract T-cell depleted hematopoietic stem cell transplantation from haploidentical donors (haplo-HSCT) has been reported to benefit from the graft-versus-leukemia effect mediated by natural killer (NK) cells when donor displays NK alloreactivity versus the recipient. NK alloreactivity is mediated by NK receptors, namely Killer Ig-like receptors (KIR) which are specific for allotypic determinants that are shared by different HLA-class I alleles (referred to as KIR ligands). It is known that KIR2DL1 recognizes HLA-C alleles characterized by Lys at position 80 (C2 group), KIR2DL2/3 recognize HLA-C alleles characterized by Asn at position 80 (C1 group), KIR3DL1 recognizes HLA-B alleles sharing the Bw4 supertypic specificity (Bw4 group) and KIR3DL2 recognizes HLA-A3 and –A11 alleles. KIR2D/3DL are inhibitory receptors that, upon engagement with the cognate ligand, inhibit lysis. Activating KIRs, highly homologous in the extracellular domain to the inhibitory counterparts, are KIR2DS1, KIR2DS2 and KIR3DS1, but only KIR2DS1 has been shown to specifically recognize C2 group of alleles expressed on B-EBV cells. We analyzed 21 children with leukemia receiving haplo-HSCT from a relative after a myeloablative conditioning regimen; in all pairs, the expression of a given KIR ligand (HLA class I allele) of the donor was missing in the patient (i.e. KIR ligand-mismatched haplo-HSCT). T-cell depletion was performed through positive selection of CD34+ cells; no pharmacological immune suppression was employed after HSCT. KIR genotype of all donors was evaluated to detect the presence of the various inhibitory and activating KIR genes. Phenotypic analyses were performed on NK cells derived from the donor and the patient at different time points after HSCT. Thanks to the availability of new mAbs able to discriminate between the inhibitory and the activating forms of a certain KIR, we could identify the alloreactive NK cell subset at the population level. These alloreactive NK cells express the KIR specific for the KIR ligand-mismatch (permissive inhibitory KIR) and the activating KIR (if present), while they do not express all inhibitory KIR specific for the patient HLA alleles and NKG2A. Thus, in most instances, we could precisely identify the size of the alloreactive NK cell subset in the donor and in the reconstituted repertoire of the recipient. Functional assays were performed to assess alloreactivity, using appropriate B-EBV cell lines and, if available, patient’s leukemia blasts. In some cases, also NK cell clones were extensively studied, for phenotype and receptor involvement in killing activity. We found that, in most transplanted patients, variable proportions of donor-derived alloreactive NK cells displaying anti-leukemia activity were generated and maintained even at late time-points after transplantation. Donor-derived KIR2DL1+ NK cells isolated from the recipient displayed the expected capability of selectively killing C1/C1 target cells, including patient leukemia blasts. Differently, KIR2DL2/3+ NK cells displayed poor alloreactivity against leukemia cells carrying HLA alleles belonging to the C2 specificity. Unexpectedly, this was due to recognition of C2 by KIR2DL2/3, as revealed by receptor blocking experiments and by binding assays of soluble KIR to HLA-C transfectants. Remarkably, however, C2/C2 leukemia blasts were killed by KIR2DL2/3+ (or by NKG2A+) NK cells that co-expressed KIR2DS1. This could be explained by the ability of KIR2DS1 to directly recognize C2 on leukemia cells. A role for the KIR2DS2 activating receptor in leukemia cell lysis could not be established. Taken together, these findings provide new information on NK alloreactivity in haplo-HSCT that may greatly impact on the selection of the optimal donor.

HLA Influence on NK Cell Expansion

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 4924-4924
Author(s):  
Jennifer Schellekens ◽  
Anna Stserbakova ◽  
Madis Tõns ◽  
Hele Everaus ◽  
Marcel GJ Tilanus ◽  
...  
Keyword(s):  
Nk Cells ◽  
Cell Lines ◽  
Cell Expansion ◽  
Nk Cell ◽  
Killer Cell ◽  
Hla Class I ◽  
Class I ◽  
Haematopoietic Stem Cell ◽  
Specific Priming

Abstract Natural Killer (NK) cells are effector cells in the innate immune system. The anti-leukaemic capacities of NK cells in haematopoietic stem cell transplantation make these cells a potential treatment modality to improve clinical outcome. Immunotherapy with NK cells requires transfusion of large quantities, which obviates the need for an in vitro culture system for NK cells. The killer cell immunoglobulin-like receptors (KIR) on NK cells recognise defined groups of HLA class I alleles. To elucidate the influence of these interactions on proliferation, the peripheral blood mononuclear cells (PBMCs) of 29 patients and donors were cultured in CellGro SCGM with IL-2 and OKT3 antibody to expand the NK cell fraction. The killer cell immunoglobulin-like receptor (KIR) and HLA repertoire were determined by sequence specific priming and sequence based typing respectively. The percentage of NK cell expansion from the total PBMC fraction varied between 5.4% and 71.6%. A significantly better NK cell expansion was observed for individuals homozygous for HLA-C epitope group 2 (p&lt;0.05). For evaluation of cytolytic competence of the cultured NK cells, specific killing of an HLA class I expression deficient LCL 721.221 cell line and three 721.221 cell lines transfected with different HLA-C alleles was determined. A significantly better NK cell-induced specific cytotoxicity was observed towards the untransfected 721.221 cells compared to the HLA-C transfected 721.221 cells. No significant differences were observed between killing of the three HLA-C transfected 721.221 cell lines. We have shown that cytolytic capacities of the cultured NK cells are maintained and in vitro expansion of NK cells is dependant on the presence of HLA-C alleles.

Natural killer (NK) cell activation, cytokine production, and cytotoxicity in human PBMC/myeloma cell co-culture exposed to elotuzumab (Elo) alone or in combination with lenalidomide (Len).

2012 ◽  
Vol 30 (15_suppl) ◽  
pp. 8087-8087 ◽  
Author(s):  
Balaji Balasa ◽  
Rui Yun ◽  
Nicole Belmar ◽  
Gary Starling ◽  
Audie Rice
Keyword(s):  
Nk Cells ◽  
Cell Activation ◽  
Nk Cell ◽  
Myeloma Cell ◽  
Cell Killing ◽  
Cell Counting ◽  
Target Cells ◽  
Human Pbmc ◽  
Myeloma Cells ◽  
Ifn Γ

8087 Background: Elo is a monoclonal IgG1 antibody targeting CS1, a cell surface glycoprotein highly expressed on >95% of myeloma cells. In preclinical models Elo exerts anti-myeloma activity via NK cell-mediated antibody-dependent cellular cytotoxicity. Len is an immunomodulatory agent that may activate NK cells. The combination of Elo + Len synergistically enhanced anti-tumor activity in myeloma xenograft models. We investigated the mechanism of enhancing NK cell activation and myeloma cell killing with Elo + Len. Methods: Human PBMC/OPM-2 co-cultures were treated for 24-72h with Elo, Len, or Elo + Len. Activation markers and adhesion receptors were evaluated by flow cytometry. Cytokines were measured by Luminex and ELISpot assays. Cytotoxicity was assessed by cell counting. Results: Elo + Len increased IFN-γ secretion significantly more than Elo or Len alone. IFN-γ elevates ICAM-1 expression, and ICAM-1 surface expression on OPM-2 target cells increased synergistically with Elo + Len. Elo, Elo + Len but not Len increased expression of CD25 (IL-2Rα) on NK cells. Len increased the levels of IL-2, but those were decreased in the presence of Elo due to increased consumption by CD25 expressing NK cells. Blocking uptake of IL-2 with anti-CD25 resulted in higher IL-2 levels than with Len. ELISpot assays confirmed that Elo + Len significantly increased the number of IL-2-producing cell colonies compared with Elo or Len. Elo induced NK dependent myeloma cell killing, and the effect was significantly higher with Elo + Len. Conclusions: Elo alone activated NK cells and mediated the killing of myeloma cells in PBMC/OPM-2 co-cultures. Elo + Len synergistically enhanced myeloma cell killing and increased expression/production of IFN-γ, ICAM-1, IL-2, and CD25. [Table: see text]

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