IFN-γ production in human NK cells through the engagement of CD8 by soluble or surface 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.

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Analysis of natural killer cells in TAP2-deficient patients: expression of functional triggering receptors and evidence for the existence of inhibitory receptor(s) that prevent lysis of normal autologous cells

Blood ◽

10.1182/blood.v99.5.1723 ◽

2002 ◽

Vol 99 (5) ◽

pp. 1723-1729 ◽

Cited By ~ 47

Author(s):

Massimo Vitale ◽

Jacques Zimmer ◽

Roberta Castriconi ◽

Daniel Hanau ◽

Lionel Donato ◽

...

Keyword(s):

Nk Cells ◽

Natural Killer ◽

Antigen Processing ◽

Nk Cell ◽

Hla Class I ◽

Surface Expression ◽

Class I ◽

Inhibitory Receptor ◽

Surface Receptors ◽

Hla Class I Molecules

Natural killer (NK) cells are characterized by the ability to kill cells that lack HLA class I molecules while sparing autologous normal (HLA class I+) cells. However, patients with transporter-associated antigen processing (TAP) deficiency, though displaying strong reductions of HLA class I surface expression, in most instances do not experience NK-mediated autoimmune phenomena. A possible mechanism by which TAP−/− NK cells avoid autoreactivity against autologous HLA class I–deficient cells could be based on either quantitative or qualitative defects of surface receptors involved in NK cell triggering. In this study we show that NK cells derived from 2 patients with TAP2−/− express normal levels of all known triggering receptors. As revealed by the analysis of polyclonal and clonal NK cells, these receptors display normal functional capabilities and allow the killing of a panel of NK-susceptible targets, including autologous B-LCLs. On the other hand, TAP2−/− NK cells were unable to kill either allogeneic (HLA class I+) or autologous (HLA class I− ) phytohemagglutinin (PHA) blasts even in the presence of anti-HLA class I monoclonal antibody. These data suggest that TAP2−/− NK cells express still unknown inhibitory receptor(s) capable of down-regulating the NK cell cytotoxicity on binding to surface ligand(s) expressed by T cell blasts. Functional analyses, both at the polyclonal and at the clonal level, are consistent with the concept that the putative inhibitory receptor is expressed by virtually all TAP2−/− NK cells, whereas it is present only in rare NK cells from healthy persons. Another possibility would be that TAP2−/− NK cells are missing a still unidentified triggering receptor involved in NK cell-mediated killing of PHA blasts.

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Genetically Modified HLA Class I Molecules Able to Inhibit Human NK Cells Without Provoking Alloreactive CD8+CTLs

The Journal of Immunology ◽

10.4049/jimmunol.168.7.3266 ◽

2002 ◽

Vol 168 (7) ◽

pp. 3266-3274 ◽

Cited By ~ 25

Author(s):

Alexandra Sharland ◽

Amy Patel ◽

Josie Han Lee ◽

Aimee E. Cestra ◽

Susan Saidman ◽

...

Keyword(s):

Nk Cells ◽

Genetically Modified ◽

Hla Class I ◽

Class I ◽

Hla Class I Molecules

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HLA Class I Molecules as Immune Checkpoints for NK Cell Alloreactivity and Anti-Viral Immunity in Kidney Transplantation

Frontiers in Immunology ◽

10.3389/fimmu.2021.680480 ◽

2021 ◽

Vol 12 ◽

Author(s):

Burcu Duygu ◽

Timo I. Olieslagers ◽

Mathijs Groeneweg ◽

Christina E. M. Voorter ◽

Lotte Wieten

Keyword(s):

Kidney Transplantation ◽

Nk Cells ◽

Cell Activation ◽

Nk Cell ◽

Hla Class I ◽

Class I ◽

Immune Checkpoints ◽

Viral Immunity ◽

Nk Cell Receptors ◽

Hla Class I Molecules

Natural killer (NK) cells are innate lymphocytes that can kill diseased- or virally-infected cells, mediate antibody dependent cytotoxicity and produce type I immune-associated cytokines upon activation. NK cells also contribute to the allo-immune response upon kidney transplantation either by promoting allograft rejection through lysis of cells of the transplanted organ or by promoting alloreactive T cells. In addition, they protect against viral infections upon transplantation which may be especially relevant in patients receiving high dose immune suppression. NK cell activation is tightly regulated through the integrated balance of signaling via inhibitory- and activating receptors. HLA class I molecules are critical regulators of NK cell activation through the interaction with inhibitory- as well as activating NK cell receptors, hence, HLA molecules act as critical immune checkpoints for NK cells. In the current review, we evaluate how NK cell alloreactivity and anti-viral immunity are regulated by NK cell receptors belonging to the KIR family and interacting with classical HLA class I molecules, or by NKG2A/C and LILRB1/KIR2DL4 engaging non-classical HLA-E or -G. In addition, we provide an overview of the methods to determine genetic variation in these receptors and their HLA ligands.

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HLA upregulation during dengue virus infection suppresses the natural killer cell response

10.1101/698803 ◽

2019 ◽

Author(s):

Julia L. McKechnie ◽

Davis Beltran ◽

Arcelys Pitti ◽

Lisseth Saenz ◽

Ana B. Araúz ◽

...

Keyword(s):

Dengue Virus ◽

Nk Cells ◽

Natural Killer ◽

Cell Response ◽

Nk Cell ◽

Hla Class I ◽

Denv Infection ◽

Class I ◽

Hla Class I Molecules

AbstractDengue virus (DENV) is the most prevalent mosquito-borne virus in the world and a major cause of morbidity in the tropics and subtropics. Upregulation of HLA class I molecules has long been considered a feature of DENV infection, yet this has not been evaluated in the setting of natural infection. Natural killer (NK) cells, an innate immune cell subset critical for mounting an early response to viral infection, are inhibited by self HLA class I, suggesting that upregulation of HLA class I during DENV infection could dampen the NK cell response. Here we addressed whether upregulation of HLA class I molecules occurs during in vivo DENV infection and, if so, whether this suppresses the NK cell response. We found that HLA class I expression was indeed upregulated during acute DENV infection across multiple cell lineages in vivo. To better understand the role of HLA class I upregulation, we infected primary human monocytes, a major target of DENV infection, in vitro. Upregulation of total HLA class I is dependent on active viral replication and is mediated in part by cytokines and other soluble factors induced by infection, while upregulation of HLA-E occurs in the presence of replication-incompetent virus. Importantly, blocking DENV-infected monocytes with a pan-HLA class I Fab nearly doubles the frequency of degranulating NK cells, while blocking HLA-E does not significantly improve the NK cell response. These findings demonstrate that upregulation of HLA class I during DENV infection suppresses the NK cell response, potentially contributing to disease pathogenesis.

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A Human Histocompatibility Leukocyte Antigen (HLA)-G–specific Receptor Expressed on All Natural Killer Cells

Journal of Experimental Medicine ◽

10.1084/jem.189.7.1093 ◽

1999 ◽

Vol 189 (7) ◽

pp. 1093-1100 ◽

Cited By ~ 506

Author(s):

Sumati Rajagopalan ◽

Eric O. Long

Keyword(s):

Nk Cells ◽

Natural Killer ◽

Killer Cell ◽

Hla Class I ◽

Class I ◽

Target Cells ◽

Trophoblast Cells ◽

Leukocyte Antigen ◽

Hla Class I Molecules ◽

Decidual Cells

Human natural killer (NK) cells express several killer cell immunoglobulin (Ig)-like receptors (KIRs) that inhibit their cytotoxicity upon recognition of human histocompatibility leukocyte antigen (HLA) class I molecules on target cells. Additional members of the KIR family, including some that deliver activation signals, have unknown ligand specificity and function. One such KIR, denoted KIR2DL4, is structurally divergent from other KIRs in the configuration of its two extracellular Ig domains and of its transmembrane and cytoplasmic domains. Here we show that recombinant soluble KIR2DL4 binds to cells expressing HLA-G but not to cells expressing other HLA class I molecules. Unlike other HLA class I–specific KIRs, which are clonally distributed on NK cells, KIR2DL4 is expressed at the surface of all NK cells. Furthermore, functional transfer of KIR2DL4 into the cell line NK-92 resulted in inhibition of lysis of target cells that express HLA-G, but not target cells that express other class I molecules including HLA-E. Therefore, given that HLA-G expression is restricted to fetal trophoblast cells, KIR2DL4 may provide important signals to maternal NK decidual cells that interact with trophoblast cells at the maternal–fetal interface during pregnancy.

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Quantitative HLA Class I Expression and KIR-Mismatch between NK-Cell-Donor and Patient Predict NK Cell Mediated Lysis of Leukemic Cells from Children with Acute Lymphatic Leukemia.

Blood ◽

10.1182/blood.v108.11.5205.5205 ◽

2006 ◽

Vol 108 (11) ◽

pp. 5205-5205

Author(s):

Matthias M. Pfeiffer ◽

Michael Schumm ◽

Klaus Dietz ◽

Tobias Feuchtinger ◽

Rupert Handgretinger ◽

...

Keyword(s):

Nk Cells ◽

Adhesion Molecules ◽

Nk Cell ◽

Hla Class I ◽

Class I ◽

Leukemic Cells ◽

Specific Lysis ◽

Lymphatic Leukemia ◽

Hla Class I Molecules ◽

The Impact

Abstract Relapses represent a major problem after transplantations in children with ALL. Natural Killer (NK) cells have been shown to exert remarkable Graft versus Leukemia effects after mismatched stem cell transplantation in myeloic leukemia, whereas the efficacy against lymphatic leukemia is still unclear. We therefore measured intensity of HLA class I expression on leukemic blasts by quantitative FACS analysis and investigated the impact of quantitative HLA class I expression, of several adhesion molecules and of KIR-mismatch on NK cell mediated lysis of the leukemic blasts from 21 pediatric patients with ALL. Expression of HLA class I molecules differed widely from patient to patient (range 5000–500000) and was reduced in comparison to B cells from healthy donors in 70% of cases. NK cells killed leukemic blasts very heterogeneously but a clear association between number of HLA class I molecules per cell and specific lysis (range 13–98%) was found (r2=0.68, p<0.0001). For the subgroup of leukemic blasts without KIR-mismatch, this association was even stronger (r2=0.98), whereas a weak association was found for the subgroup with KIR-mismatch, since most of these targets were lysed more efficiently than one could expect according to HLA class I expression alone. KIR-mismatch alone (t-test, p=0.45) as well as different patterns of adhesion molecules (ICAM1-3, LFA1/3) and CD95 had no significant influence. However, a multivariate model taking both HLA expression and KIR-ligand-mismatch into account, provided an even stronger association (r2=0.87 p<0.0001) for the whole group. Lysis was mainly dependent on HLA class I expression and in addition on KIR-mismatch for these leukemic cells. Assessment of HLA expression on leukemic blasts and KIR-receptor-ligand-mismatch between donor and recipient may be valuable to define patients who will benefit most from a NK mediated GvL effect.

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HLA Class I Knockout Converts Allogeneic Primary NK Cells Into Suitable Effectors for “Off-the-Shelf” Immunotherapy

Frontiers in Immunology ◽

10.3389/fimmu.2020.586168 ◽

2021 ◽

Vol 11 ◽

Author(s):

Keven Hoerster ◽

Markus Uhrberg ◽

Constanze Wiek ◽

Peter A. Horn ◽

Helmut Hanenberg ◽

...

Keyword(s):

T Cells ◽

Nk Cells ◽

Genome Editing ◽

Nk Cell ◽

Human Cancer ◽

Hla Class I ◽

Class I ◽

Cellular Immunotherapy ◽

Single Chain ◽

Hla Class I Molecules

Cellular immunotherapy using chimeric antigen receptors (CARs) so far has almost exclusively used autologous peripheral blood-derived T cells as immune effector cells. However, harvesting sufficient numbers of T cells is often challenging in heavily pre-treated patients with malignancies and perturbed hematopoiesis and perturbed hematopoiesis. Also, such a CAR product will always be specific for the individual patient. In contrast, NK cell infusions can be performed in non-HLA-matched settings due to the absence of alloreactivity of these innate immune cells. Still, the infused NK cells are subject to recognition and rejection by the patient’s immune system, thereby limiting their life-span in vivo and undermining the possibility for multiple infusions. Here, we designed genome editing and advanced lentiviral transduction protocols to render primary human NK cells unsusceptible/resistant to an allogeneic response by the recipient’s CD8+ T cells. After knocking-out surface expression of HLA class I molecules by targeting the B2M gene via CRISPR/Cas9, we also co-expressed a single-chain HLA-E molecule, thereby preventing NK cell fratricide of B2M-knockout (KO) cells via “missing self”-induced lysis. Importantly, these genetically engineered NK cells were functionally indistinguishable from their unmodified counterparts with regard to their phenotype and their natural cytotoxicity towards different AML cell lines. In co-culture assays, B2M-KO NK cells neither induced immune responses of allogeneic T cells nor re-activated allogeneic T cells which had been expanded/primed using irradiated PBMNCs of the respective NK cell donor. Our study demonstrates the feasibility of genome editing in primary allogeneic NK cells to diminish their recognition and killing by mismatched T cells and is an important prerequisite for using non-HLA-matched primary human NK cells as readily available, “off-the-shelf” immune effectors for a variety of immunotherapy indications in human cancer.

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