Human Natural Killer Cells Achieve Tolerance by Preferentially Endowing Functional Competence to Inhibitory Killer Ig-Like Receptors Specific for Self-HLA Class I.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 919-919
Author(s):  
Junli Yu ◽  
Clara Pinto-Agnello ◽  
Joseph Chewning ◽  
Katharine C. Hsu
Keyword(s):  
Nk Cells ◽  
Natural Killer ◽  
Class I ◽  
Target Cells ◽  
Inhibitory Receptors ◽  
Hla Ligand ◽  
Self Tolerance ◽  
Hla Ligands ◽  
Ifn Γ ◽  
Haplotype A

Abstract Natural killer (NK) cells are capable of cytotoxic targeting of virally infected cells and some tumor cells. It has been well-demonstrated that NK cells recognize target cells that have down-regulated MHC class I antigen expression (i.e. “missing self recognition”), and that it is the lack of class I engagement of inhibitory receptors such as the killer Ig-like receptors (KIR) that thereby allows NK activation and effector function. How these same inhibitory receptors achieve self-tolerance and simultaneously avoid autoimmunity in humans has not been clear, as more than 60% of individuals have inhibitory KIR for which they lack the HLA ligand. We demonstrate that mature NK cells achieve self-tolerance by preferentially endowing functional competence to the inhibitory KIRs for which they exhibit the cognate HLA ligands. To allow evaluation of inhibitory KIR and avoid interference from potentially class-I recognizing activating KIR, we analyzed NK cells from 10 individuals with various HLA backgrounds, but who were all homozygous for KIR haplotype-A. KIR haplotype-A contains the inhibitory KIR receptors 2DL3, 2DL1, and 3DL1, specific for HLA-Cw3, -Cw4, and -Bw4 ligands respectively, in addition to at most one other activating KIR whose ligand is unknown. Using 6-color staining and flow cytometric analysis of intracellular IFN-γ production, we evaluated the responsiveness of 30 inhibitory KIR-expressing NK subsets following activation with 721.221, a target cell line deficient in class I expression, with 721.221 transfectants expressing HLA-Cw3, -Cw4, or -Bw4 ligands, and with B-lymphocyte cell lines with diverse HLA phenotypes. NK cells exclusively expressing an inhibitory KIR for self-HLA demonstrated increased IFN-γ when coincubated with target cells lacking the cognate HLA ligand, whereas NK cells exclusively expressing an inhibitory KIR for non-self HLA were hyporesponsive to all targets. In all individuals, NK cells expressing inhibitory KIR specific for self-HLA were significantly more responsive than NK cells expressing inhibitory KIR for non-self HLA (p<0.001). All 3 inhibitory KIR (KIR2DL3, 2DL1, and 3DL1) demonstrated capacity for tolerance, with predictable response patterns based on the HLA background of the individual. NK cells lacking all inhibitory NK receptors (KIR−CD94/NKG2A−ILT2−) were identifiable and were markedly hyporesponsive. KIR−CD94/NKG2A+ILT2+ NK cells were also minimally responsive, comparable to the NK subset expressing inhibitory KIR for non-self HLA. These results demonstrate NK tolerance in humans, consistent with the licensing model proposed in mice: NK cells expressing inhibitory KIR to self-HLA are significantly more responsive than NK cells expressing inhibitory KIR for non-self HLA, and are rendered tolerant to self through inhibition upon binding to self-HLA ligands. NK cells expressing inhibitory KIR for non-self HLA are hyporesponsive and therefore rendered tolerant and incapable of autoreactivity when ligand is not engaged.

scholarly journals Inhibitory Receptors Alter Natural Killer Cell Interactions with Target Cells Yet Allow Simultaneous Killing of Susceptible Targets

1999 ◽  
Vol 190 (7) ◽  
pp. 1005-1012 ◽  
Author(s):  
Mikael Eriksson ◽  
Guenther Leitz ◽  
Erik Fällman ◽  
Ove Axner ◽  
James C. Ryan ◽  
...  
Keyword(s):  
Nk Cells ◽  
Natural Killer ◽  
Mhc Class I ◽  
Optical Tweezers ◽  
Target Cell ◽  
Nk Cell ◽  
Killer Cell ◽  
Class I ◽  
Target Cells ◽  
Inhibitory Receptors

Inhibitory receptors expressed on natural killer (NK) cells abrogate positive signals upon binding corresponding major histocompatibility complex (MHC) class I molecules on various target cells. By directly micromanipulating the effector–target cell encounter using an optical tweezers system which allowed temporal and spatial control, we demonstrate that Ly49–MHC class I interactions prevent characteristic cellular responses in NK cells upon binding to target cells. Furthermore, using this system, we directly demonstrate that an NK cell already bound to a resistant target cell may simultaneously bind and kill a susceptible target cell. Thus, although Ly49-mediated inhibitory signals can prevent many types of effector responses, they do not globally inhibit cellular function, but rather the inhibitory signal is spatially restricted towards resistant targets.

scholarly journals A subset of natural killer cells achieves self-tolerance without expressing inhibitory receptors specific for self-MHC molecules

Blood ◽  
2005 ◽  
Vol 105 (11) ◽  
pp. 4416-4423 ◽  
Author(s):  
Nadine C. Fernandez ◽  
Emmanuel Treiner ◽  
Russell E. Vance ◽  
Amanda M. Jamieson ◽  
Suzanne Lemieux ◽  
...  
Keyword(s):  
Nk Cells ◽  
Natural Killer ◽  
Nk Cell ◽  
Class I ◽  
Inhibitory Receptor ◽  
Inhibitory Receptors ◽  
Mhc Molecules ◽  
Histocompatibility Complex ◽  
Mhc Class I Molecule ◽  
Self Tolerance

Abstract It is widely believed that self-tolerance of natural killer (NK) cells occurs because each NK cell expresses at least one inhibitory receptor specific for a host major histocompatibility complex (MHC) class I molecule. Here we report that some NK cells lack all known self-MHC–specific inhibitory receptors, yet are nevertheless self-tolerant. These NK cells exhibit a normal cell surface phenotype and some functional activity. However, they respond poorly to class I–deficient normal cells, tumor cells, or cross-linking of stimulatory receptors, suggesting that self-tolerance is established by dampening stimulatory signaling. Thus, self-tolerance of NK cells in normal animals can occur independently of MHC-mediated inhibition, and hyporesponsiveness plays a role in self-tolerance of NK cells, as also proposed for B and T cells.

scholarly journals Recognition of virus-infected cells by natural killer cell clones is controlled by polymorphic target cell elements.

1993 ◽  
Vol 178 (3) ◽  
pp. 961-969 ◽  
Author(s):  
M S Malnati ◽  
P Lusso ◽  
E Ciccone ◽  
A Moretta ◽  
L Moretta ◽  
...  
Keyword(s):  
T Cells ◽  
Nk Cells ◽  
Natural Killer ◽  
Target Cell ◽  
Nk Cell ◽  
Class I ◽  
Target Cells ◽  
Cell Recognition ◽  
Infected Cells ◽  
Nk Cell Recognition

Natural killer (NK) cells provide a first line of defense against viral infections. The mechanisms by which NK cells recognize and eliminate infected cells are still largely unknown. To test whether target cell elements contribute to NK cell recognition of virus-infected cells, human NK cells were cloned from two unrelated donors and assayed for their ability to kill normal autologous or allogeneic cells before and after infection by human herpesvirus 6 (HHV-6), a T-lymphotropic herpesvirus. Of 132 NK clones isolated from donor 1, all displayed strong cytolytic activity against the NK-sensitive cell line K562, none killed uninfected autologous T cells, and 65 (49%) killed autologous T cells infected with HHV-6. A panel of representative NK clones from donors 1 and 2 was tested on targets obtained from four donors. A wide heterogeneity was observed in the specificity of lysis of infected target cells among the NK clones. Some clones killed none, some killed only one, and others killed more than one of the different HHV-6-infected target cells. Killing of infected targets was not due to complete absence of class I molecules because class I surface levels were only partially affected by HHV-6 infection. Thus, target cell recognition is not controlled by the effector NK cell alone, but also by polymorphic elements on the target cell that restrict NK cell recognition. Furthermore, NK clones from different donors display a variable range of specificities in their recognition of infected target cells.

scholarly journals The Ly-49D Receptor Activates Murine Natural Killer Cells

1996 ◽  
Vol 184 (6) ◽  
pp. 2119-2128 ◽  
Author(s):  
L.H. Mason ◽  
S.K. Anderson ◽  
W.M. Yokoyama ◽  
H.R.C. Smith ◽  
R. Winkler-Pickett ◽  
...  
Keyword(s):  
Gene Family ◽  
Nk Cells ◽  
Natural Killer ◽  
Cell Function ◽  
Nk Cell ◽  
Cell Subset ◽  
Class I ◽  
Target Cells ◽  
Color Flow ◽  
Functional Capabilities

Proteins encoded by members of the Ly-49 gene family are predominantly expressed on murine natural killer (NK) cells. Several members of this gene family have been demonstrated to inhibit NK cell lysis upon recognizing their class I ligands on target cells. In this report, we present data supporting that not all Ly-49 proteins inhibit NK cell function. Our laboratory has generated and characterized a monoclonal antibody (mAb) (12A8) that can be used to recognize the Ly-49D subset of murine NK cells. Transfection of Cos-7 cells with known members of the Ly-49 gene family revealed that 12A8 recognizes Ly-49D, but also cross-reacts with the Ly-49A protein on B6 NK cells. In addition, 12A8 demonstrates reactivity by both immunoprecipitation and two-color flow cytometry analysis with an NK cell subset that is distinct from those expressing Ly-49A, C, or G2. An Ly-49D+ subset of NK cells that did not express Ly49A, C, and G2 was isolated and examined for their functional capabilities. Tumor targets and concanovalin A (ConA) lymphoblasts from a variety of H2 haplotypes were examined for their susceptibility to lysis by Ly-49D+ NK cells. None of the major histocompatibility complex class I–bearing targets inhibited lysis of Ly-49D+ NK cells. More importantly, we demonstrate that the addition of mAb 12A8 to Ly-49D+ NK cells can augment lysis of FcγR+ target cells in a reverse antibody-dependent cellular cytotoxicity–type assay and induces apoptosis in Ly49D+ NK cells. Furthermore, the cytoplasmic domain of Ly-49D does not contain the V/IxYxxL immunoreceptor tyrosine-based inhibitory motif found in Ly-49A, C, or G2 that has been characterized in the human p58 killer inhibitory receptors. Therefore, Ly-49D is the first member of the Ly-49 family characterized as transmitting positive signals to NK cells, rather than inhibiting NK cell function.

scholarly journals Slaying the Trojan Horse: Natural Killer Cells Exhibit Robust Anti-HIV-1 Antibody-Dependent Activation and Cytolysis against Allogeneic T Cells

2014 ◽  
Vol 89 (1) ◽  
pp. 97-109 ◽  
Author(s):  
Shayarana L. Gooneratne ◽  
Jonathan Richard ◽  
Wen Shi Lee ◽  
Andrés Finzi ◽  
Stephen J. Kent ◽  
...  
Keyword(s):  
T Cells ◽  
Nk Cells ◽  
Natural Killer ◽  
Nk Cell ◽  
Target Cells ◽  
Dependent Manner ◽  
Inhibitory Receptors ◽  
Cell Responses ◽  
Anti Hiv ◽  
Hiv 1

ABSTRACTMany attempts to design prophylactic human immunodeficiency virus type 1 (HIV-1) vaccines have focused on the induction of neutralizing antibodies (Abs) that block infection by free virions. Despite the focus on viral particles, virus-infected cells, which can be found within mucosal secretions, are more infectious than free virus bothin vitroandin vivo. Furthermore, assessment of human transmission couples suggests infected seminal lymphocytes might be responsible for a proportion of HIV-1 transmissions. Although vaccines that induce neutralizing Abs are sought, only some broadly neutralizing Abs efficiently block cell-to-cell transmission of HIV-1. As HIV-1 vaccines need to elicit immune responses capable of controlling both free and cell-associated virus, we evaluated the potential of natural killer (NK) cells to respond in an Ab-dependent manner to allogeneic T cells bearing HIV-1 antigens. This study presents data measuring Ab-dependent anti-HIV-1 NK cell responses to primary and transformed allogeneic T-cell targets. We found that NK cells are robustly activated in an anti-HIV-1 Ab-dependent manner against allogeneic targets and that tested target cells are subject to Ab-dependent cytolysis. Furthermore, the educated KIR3DL1+NK cell subset from HLA-Bw4+individuals exhibits an activation advantage over the KIR3DL1−subset that contains both NK cells educated through other receptor/ligand combinations and uneducated NK cells. These results are intriguing and important for understanding the regulation of Ab-dependent NK cell responses and are potentially valuable for designing Ab-dependent therapies and/or vaccines.IMPORTANCENK cell-mediated anti-HIV-1 antibody-dependent functions have been associated with protection from infection and disease progression; however, their role in protecting from infection with allogeneic cells infected with HIV-1 is unknown. We found that HIV-1-specific ADCC antibodies bound to allogeneic cells infected with HIV-1 or coated with HIV-1 gp120 were capable of activating NK cells and/or trigging cytolysis of the allogeneic target cells. This suggests ADCC may be able to assist in preventing infection with cell-associated HIV-1. In order to fully utilize NK cell-mediated Ab-dependent effector functions, it might also be important that educated NK cells, which hold the highest activation potential, can become activated against targets bearing HIV-1 antigens and expressing the ligands for self-inhibitory receptors. Here, we show that with Ab-dependent stimulation, NK cells expressing inhibitory receptors can mediate robust activation against targets expressing the ligands for those receptors.

scholarly journals MHC class I–deficient natural killer cells acquire a licensed phenotype after transfer into an MHC class I–sufficient environment

2010 ◽  
Vol 207 (10) ◽  
pp. 2073-2079 ◽  
Author(s):  
Julie M. Elliott ◽  
Joseph A. Wahle ◽  
Wayne M. Yokoyama
Keyword(s):  
Bone Marrow ◽  
Nk Cells ◽  
Natural Killer ◽  
Mhc Class I ◽  
Interferon Γ ◽  
Class I ◽  
Target Cells ◽  
Major Histocompatability Complex ◽  
Gain Of Function ◽  
Mhc Class I Molecule

In MHC class I–deficient hosts, natural killer (NK) cells are hyporesponsive to cross-linking of activation receptors. Functional competence requires engagement of a self–major histocompatability complex (MHC) class I–specific inhibitory receptor, a process referred to as “licensing.” We previously suggested that licensing is developmentally determined in the bone marrow. In this study, we find that unlicensed mature MHC class I–deficient splenic NK cells show gain-of-function and acquire a licensed phenotype after adoptive transfer into wild-type (WT) hosts. Transferred NK cells produce WT levels of interferon-γ after engagement of multiple activation receptors, and degranulate at levels equivalent to WT NK cells upon coincubation with target cells. Only NK cells expressing an inhibitory Ly49 receptor specific for a cognate host MHC class I molecule show this gain-of-function. Therefore, these findings, which may be relevant to clinical bone marrow transplantation, suggest that neither exposure to MHC class I ligands during NK development in the BM nor endogenous MHC class I expression by NK cells themselves is absolutely required for licensing.

scholarly journals Heterogeneous phenotypes of expression of the NKB1 natural killer cell class I receptor among individuals of different human histocompatibility leukocyte antigens types appear genetically regulated, but not linked to major histocompatibililty complex haplotype.

1996 ◽  
Vol 183 (4) ◽  
pp. 1817-1827 ◽  
Author(s):  
J E Gumperz ◽  
N M Valiante ◽  
P Parham ◽  
L L Lanier ◽  
D Tyan
Keyword(s):  
T Cells ◽  
Cell Surface ◽  
Nk Cells ◽  
Natural Killer ◽  
Killer Cell ◽  
Peripheral Blood Lymphocytes ◽  
Class I ◽  
Cell Surface Expression ◽  
Target Cells ◽  
Hla Type

Natural killer (NK) cells that express the NKB1 receptor are inhibited from killing target cells that possess human histocompatibility leukocyte antigen (HLA) B molecules bearing the Bw4 serological epitope. To investigate whether NKB1 expression is affected by HLA type, peripheral blood lymphocytes of 203 HLA-typed donors were examined. Most donors had a single population of NKB1+ cells, but some had two populations expressing different cell surface levels of NKB1, and others had no detectable NKB1+ cells. Among the donors expressing NKB1, both the relative abundance of NKB1+ NK cells and their level of cell surface expression varied substantially. The percentage of NKB1+ NK cells ranged from 0 to &gt;75% (mean 14.7%), and the mean fluorescence of the positive population varied over three orders of magnitude. For each donor, the small percentage of T cells expressing NKB1 (usually &lt;2%), had a pattern of expression mirroring that of the NK cells. NKB1 expression by NK and T cells remained stable over the 2-yr period that five donors were tested. Patterns of NKB1 expression were not associated with Bw4 or Bw6 serotype of the donor or with the presence of any individual HLA-A or -B antigens. Cells expressing NKB1 are often found in donors who do not possess an appropriate class I ligand, and can be absent in those who express Bw4+ HLA-B antigens. Family studies further suggested that the phenotype of NKB1 expression is inherited but not HLA linked. Whereas identical twins show matching patterns of NKB1 expression, HLA-identical siblings can differ in NKB1 expression, and conversely, HLA-disparate siblings can be similar. Thus NKB1 expression phenotypes are tightly regulated and extremely heterogeneous, but not correlated with HLA type.

scholarly journals The Bw4 public epitope of HLA-B molecules confers reactivity with natural killer cell clones that express NKB1, a putative HLA receptor.

1995 ◽  
Vol 181 (3) ◽  
pp. 1133-1144 ◽  
Author(s):  
J E Gumperz ◽  
V Litwin ◽  
J H Phillips ◽  
L L Lanier ◽  
P Parham
Keyword(s):  
Monoclonal Antibody ◽  
Nk Cells ◽  
Natural Killer ◽  
Nk Cell ◽  
Killer Cell ◽  
Inhibitory Effect ◽  
Glycosylation Site ◽  
Class I ◽  
Target Cells ◽  
Cell Clones

Although inhibition of natural killer (NK) cell-mediated lysis by the class I HLA molecules of target cells is an established phenomenon, knowledge of the features of class I molecules which induce this effect remains rudimentary. Using class I alleles HLA-B*1502 and B*1513 which differ only at residues 77-83 which define the Bw4 and Bw6 serological epitopes, we tested the hypothesis that the presence of the Bw4 epitope on class I molecules determines recognition by NKB1+ NK cells. HLA-B*1513 possesses the Bw4 epitope, whereas B*1502 has the Bw6 epitope. Lysis by NKB1+ NK cell clones of transfected target cells expressing B*1513 as the only HLA-A, -B, or -C molecule was inhibited, whereas killing of transfectants expressing B*1502 was not. Addition of an an anti-NKB1 monoclonal antibody reconstituted lysis of the targets expressing B*1513, but did not affect killing of targets bearing B*1502. The inhibitory effect of B*1513 could be similarly prevented by the addition of an anti-class I monoclonal antibody. These results show that the presence of the Bw4 epitope influences recognition of HLA-B molecules by NK cells that express NKB1, and suggest that the NKB1 molecule may act as a receptor for Bw4+ HLA-B alleles. Sequences outside of the Bw4 region must also affect recognition by NKB1+ NK cells, because lysis of transfectants expressing HLA-A*2403 or A*2501, which possess the Bw4 epitope but are in other ways substantially different from HLA-B molecules, was not increased by addition of the anti-NKB1 antibody. Asparagine 86, the single site of N-linked glycosylation on class I molecules, is in close proximity to the Bw4/Bw6 region. The glycosylation site of the Bw4-positive molecule B*5801 was mutated, and the mutant molecules tested for inhibition of NKB1+ NK cells. Inhibition that could be reversed by addition of the anti-NKB1 monoclonal antibody was observed, showing the presence of the carbohydrate moiety is not essential for class I recognition by NKB1+ NK cell clones.

scholarly journals Human natural killer cell receptors for HLA-class I molecules. Evidence that the Kp43 (CD94) molecule functions as receptor for HLA-B alleles.

1994 ◽  
Vol 180 (2) ◽  
pp. 545-555 ◽  
Author(s):  
A Moretta ◽  
M Vitale ◽  
S Sivori ◽  
C Bottino ◽  
L Morelli ◽  
...  
Keyword(s):  
Nk Cells ◽  
Natural Killer ◽  
Killer Cell ◽  
Hla Class I ◽  
Cytolytic Activity ◽  
Class I ◽  
Target Cells ◽  
Human Natural Killer Cell ◽  
Ligand Interaction ◽  
Hla Class I Molecules

GL183 or EB6 (p58) molecules have been shown to function as receptors for different HLA-C alleles and to deliver an inhibitory signal to natural killer (NK) cells, thus preventing lysis of target cells. In this study, we analyzed a subset of NK cells characterized by a p58-negative surface phenotype. We show that p58-negative clones, although specific for class I molecules do not recognize HLA-C alleles. In addition, by the use of appropriate target cells transfected with different HLA-class I alleles we identified HLA-B7 as the protective element recognized by a fraction of p58-negative clones. In an attempt to identify the receptor molecules expressed by HLA-B7-specific clones, monoclonal antibodies (mAbs) were selected after mice immunization with such clones. Two of these mAbs, termed XA-88 and XA-185, and their F(ab')2 fragments, were found to reconstitute lysis of B7+ target cells by B7-specific NK clones. Both mAbs were shown to be directed against the recently clustered Kp43 molecule (CD94). Thus, mAb-mediated masking of Kp43 molecules interferes with recognition of HLA-B7 and results in target cell lysis. Moreover, in a redirected killing assay, the cross-linking of Kp43 molecules mediated by the XA185 mAb strongly inhibited the cytolytic activity of HLA-B7-specific NK clones, thus mimicking the functional effect of B7 molecules. Taken together, these data strongly suggest that Kp43 molecules function as receptors for HLA-B7 and that this receptor/ligand interaction results in inhibition of the NK-mediated cytolytic activity. Indirect immunofluorescence and FACS analysis of a large number of random NK clones showed that Kp43 molecules (a) were brightly expressed on a subset of p58-negative clones, corresponding to those specific for HLA-B7; (b) displayed a medium/low fluorescence in the p58-negative clones that are not B7-specific as well as in most p58+ NK clones; and (c) were brightly expressed as in the p58+ clone ET34 (GL183-/EB6+, Cw4-specific). Functional analysis revealed that Kp43 functioned as an inhibitory receptor only in NK clones displaying bright fluorescence. These studies also indicate that some NK clones (e.g., the ET34) can coexpress two distinct receptors (p58 and Kp43) for different class I alleles (Cw4 and B7). Finally, we show that Kp43 molecules function as receptors only for some HLA-B alleles and that still undefined receptor(s) must exist for other HLA-B alleles including B27.

Haploidentical Natural Killer (NK) Cells Expanding In Vivo After Adoptive Transfer Exhibit Hyperfunction That Partially Overcomes Self Tolerance and Leads to Clearance of Refractory Leukemia

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 355-355 ◽  
Author(s):  
Sarah Cooley ◽  
Bree Foley ◽  
Michael R Verneris ◽  
David McKenna ◽  
Xianghua Luo ◽  
...  
Keyword(s):  
Nk Cells ◽  
Natural Killer ◽  
Adoptive Transfer ◽  
Cell Expansion ◽  
Nk Cell ◽  
Color Flow ◽  
Self Tolerance ◽  
Hla Ligands ◽  
Better Than

Abstract Abstract 355FN2 We have previously shown that adoptive transfer of haploidentical natural killer (NK) cells can induce remissions in patients with refractory or relapsed acute myeloid leukemia (AML). We hypothesize that in vivo expansion of functional NK cells is required for therapeutic efficacy. To achieve the adequate host immune suppression required for expansion we added total body irradiation (TBI) to our lymphodepleting chemotherapy regimen, giving patients fludarabine (Flu) 25 mg/m2 × 5 days, cyclophosphamide (Cy) 60 mg/kg × 2 days, and 400 cGy of TBI. The NK cell product, a CD3- and CD19-depleted lymphapheresis from a haploidentical related donor, was incubated overnight in 1000 U/ml IL-2 and infused followed by 6 doses of alternate day subcutaneous IL-2 (10 million units) to promote in vivo expansion. Because of the increased myelosuppression following the TBI, a CD34-selected filgrastim-mobilized peripheral blood graft from the same donor (target dose >3 × 106 CD34 cells/kg) was given for hematopoietic rescue. Successful in vivo NK cell expansion was prospectively defined as >100 donor-derived NK cells/ml at 14 days after adoptive transfer (by analysis of STR chimerism, % NK and the clinical absolute lymphocyte count). In the 38 evaluable patients, robust in vivo expansion was induced in 50% (absolute donor NK count of 666 ± 134 cells/μL blood), a rate considerably higher than the 10% we observed in a cohort receiving Cy/Flu alone without TBI. Successful NK cell expansion correlated with leukemia clearance (<1% marrow blasts 14 days after NK cell infusion) and remission (leukemia free with donor neutrophil engraftment at day +30; 42 days after NK infusion). Of the 19 patients who achieved NK cell expansion, 89% cleared their leukemia compared to 42% of the non-expanders (p=0.002); and 84% achieved remission vs. 10% of non-expanders (p <.0001). The robust in vivo expansion of adoptively transferred NK cells gave us the unique opportunity to study their function. We studied blood collected from patients 14 days after NK cell infusion and compared it to paired donor samples obtained at steady state from the apheresis products prior to IL-2 stimulation. Using multi-color flow cytometry, we measured CD107a expression (a surrogate marker for NK cell cytotoxicity) on NK cells which we could subset by expression of single inhibitory killer cell immunoglobulin-like receptors (KIR) (CD158a, CD158b and CD158e) or NKG2A. We defined NK subsets as self-KIR+ or non-self KIR+ based on the cognate HLA ligands (C2, C1, Bw4) present in the donor or recipient. The bulk population of in vivo expanded donor NK cells exhibited hyperfunction with 62.4±4.4% degranulation in response to class I negative K562 targets compared to 36.6±3.0% in the donor product samples (N=15; p=0.0043). As expected, the most potent NK cells in the unstimulated donor product were the self-KIR+ subset, which expressed 39.5±3.0% CD107a after incubation with K562 (N=23) compared to either the non-self KIR+subset (13.1±4.0%, N=6; p=0.0001), or the uneducated KIR−/NKG2A− subset (12.4±5.8%, N=10; p<0.0001). Remarkably, all 3 subsets exhibited even greater degranulation activity after 14 days of in vivo expansion where they were exposed to homeostatic factors and the IL-2 administered to the patient. While all subsets expressed more CD107a, the rules of education were maintained. The subset expressing self-KIR that recognized donor HLA ligands degranulated significantly better than the non-self KIR+ subset (53.5±14.1% vs. 34.3±13.6%, p<0.01). Interestingly, the in vivo expanded NK cells with KIR recognizing cognate ligands unique to the recipient also functioned better (53.1±14.3% [recipient self KIR+] vs. 32.4±12.0% [non-self KIR+], N=25 and N=6; p<0.0045), showing that the education status of adult NK cells is dynamic, not fixed. Importantly, the KIR−/NKG2A− subset functioned better after in vivo expansion (39.5±115.3%, N=12), demonstrating that adoptively transferred NK cells can acquire function by two separate mechanisms: 1) acquisition of function through NK cell education; and 2) acquisition of function by homeostatic expansion and cytokine activation. These data suggest that while hyperfunctioning NK cells that expand in vivo after adoptive transfer partially overcome self tolerance, which may augment their anti-leukemic effects, they still follow the rules of NK cell education where self KIR+ cells kill better than non-self KIR+ cells. Disclosures: No relevant conflicts of interest to declare.

Sign in / Sign up

Export Citation Format

Share Document