Missing Self Research Articles

Transfer of MHC Class I by Coating Platelets Enables Tumor Cells to Evade NK Cell Immune Surveillance by Conferring a “Pseudo Self” Phenotype

AbstractAbstract 583NK cells are a central component of the cytotoxic lymphocyte compartment capable of lysing tumor cells without prior immune sensitization of the host. The mechanisms leading to activation of NK reactivity are described by the principles of ‘missing-self’ and ‘induced-self’, which imply that cells with a low or absent expression of MHC class I ('missing-self') and/or a stress-induced expression of ligands of activating NK receptors like e.g. NKG2D ('induced-self') are preferentially recognized and eliminated by NK cells. Thus, a balance of various activating and inhibitory signals determines whether NK cell responses are initiated or not. Tumor cells often downregulate expression of MHC class I to evade T cell-mediated immune surveillance, which results in enhanced NK susceptibility. Besides the direct interaction with their target cells, NK activity is further influenced by the reciprocal interplay with various other hematopoietic cells. We and others demonstrated previously that thrombocytopenia inhibits metastasis in murine models, which is reversed by additional depletion of NK cells (e.g., Jin et al., Nature Med. 2006, Palumbo et al., Blood 2005). However, the mechanisms by which platelets impair NK-tumor interaction are largely unclear, especially in humans. Recently we reported that platelets release TGF-β upon interaction with tumor cells causing downregulation of NKG2D on NK cells, which impairs anti-tumor immunity by disturbing the principle of “induced self” (Kopp et al., Cancer Res. 2009). Here we demonstrate that platelets further enable tumor cells to evade NK cell immune surveillance by preventing detection of “missing self”: We found that tumor cells rapidly get coated in the presence of platelets, the latter expressing large amounts of MHC class I on their surface. In case of MHC class I-negative or -low cancer cells, this process results in MHC class I “pseudoexpression” on the tumor cell surface as revealed by flow cytometry, immunofluorescent staining, and electron microscopy. Platelet-derived MHC class I was found to inhibit the reactivity of autologous NK, both upon activation with cytokines and, most importantly, in cultures with platelet-coated tumor cells. Using constitutively MHC class I-negative/low tumor cells we found that blocking MHC class I restored NK cytotoxicity and IFN-γ production against platelet-coated tumor cells, but did not alter NK reactivity against the tumor cells in the absence of coating platelets. Taken together, our data indicate that platelets enable a molecular mimicry of tumor cells, allowing the latter to downregulate MHC class I in order to escape T cell immunity without inducing sufficient NK tumor immune surveillance due to conferred platelet-mediated “pseudo self”. Disclosures:No relevant conflicts of interest to declare.

Decoration of T-independent antigen with ligands for CD22 and Siglec-G can suppress immunity and induce B cell tolerance in vivo

Autoreactive B lymphocytes first encountering self-antigens in peripheral tissues are normally regulated by induction of anergy or apoptosis. According to the “two-signal” model, antigen recognition alone should render B cells tolerant unless T cell help or inflammatory signals such as lipopolysaccharide are provided. However, no such signals seem necessary for responses to T-independent type 2 (TI-2) antigens, which are multimeric antigens lacking T cell epitopes and Toll-like receptor ligands. How then do mature B cells avoid making a TI-2–like response to multimeric self-antigens? We present evidence that TI-2 antigens decorated with ligands of inhibitory sialic acid–binding Ig-like lectins (siglecs) are poorly immunogenic and can induce tolerance to subsequent challenge with immunogenic antigen. Two siglecs, CD22 and Siglec-G, contributed to tolerance induction, preventing plasma cell differentiation or survival. Although mutations in CD22 and its signaling machinery have been associated with dysregulated B cell development and autoantibody production, previous analyses failed to identify a tolerance defect in antigen-specific mutant B cells. Our results support a role for siglecs in B cell self-/nonself-discrimination, namely suppressing responses to self-associated antigens while permitting rapid “missing self”–responses to unsialylated multimeric antigens. The results suggest use of siglec ligand antigen constructs as an approach for inducing tolerance.

Natural Killer Cells: Tolerance to Self and Innate Immunity to Viral Infection and Malignancy

Natural killer (NK) cells are lymphocytes whose ability to identify and kill virally infected and malignant cells while sparing normal cells was poorly understood until the late 1980’s and the introduction of the “missing self’ hypothesis. According to this hypothesis, downregulation of major histocompatibility complex (MHC) class I molecules during viral infection or malignant transformation triggers NK activation (1). Since this hypothesis was first proposed, much has been learned about NK cell surface receptors, their role in the molecular basis of missing-self recognition, and the mechanisms underlying NK cell tolerance. In this review, we will discuss these mechanisms, as well as their relevance to viral infection and tumor immunity and stem cell transplantation.

Structure of Natural Killer Cell Receptor KLRG1 Bound to E-Cadherin Reveals Basis for MHC-Independent Missing Self Recognition

The cytolytic activity of natural killer (NK) cells is regulated by inhibitory receptors that detect the absence of self molecules on target cells. Structural studies of missing self recognition have focused on NK receptors that bind MHC. However, NK cells also possess inhibitory receptors specific for non-MHC ligands, notably cadherins, which are downregulated in metastatic tumors. We determined the structure of killer cell lectin-like receptor G1 (KLRG1) in complex with E-cadherin. KLRG1 mediates missing self recognition by binding to a highly conserved site on classical cadherins, enabling it to monitor expression of several cadherins (E-, N-, and R-) on target cells. This site overlaps the site responsible for cell-cell adhesion but is distinct from the integrin alpha(E)beta(7) binding site. We propose that E-cadherin may coengage KLRG1 and alpha(E)beta(7) and that KLRG1 overcomes its exceptionally weak affinity for cadherins through multipoint attachment to target cells, resulting in inhibitory signaling.

The strength of inhibitory input during education quantitatively tunes the functional responsiveness of individual natural killer cells

Natural killer (NK) cells express inhibitory receptors for major histocompatibility complex (MHC) class I. If self-MHC is down-regulated or absent, lack of inhibition triggers "missing self" killing. NK cells developing in the absence of MHC class I are hypo-responsive, demonstrating that MHC class I molecules are required for NK-cell education. Here, we show that the number and the type of MHC class I alleles that are present during NK-cell education quantitatively determine the frequency of responding NK cells, the number of effector functions in individual NK cells, and the amount of interferon-gamma production in NK cells of specific Ly49 subsets. A relationship between the extent of inhibitory signals during education and functional responsiveness was corroborated by an enhanced probability of NK cells expressing more than one inhibitory receptor for a single host self-MHC class I allele to degranulate after activation. Our data suggest that the capacity of an individual NK cell to respond to stimulation is quantitatively controlled by the extent of inhibitory signals that are received from MHC class I molecules during NK-cell education.

Cutting Edge: Killer Ig-Like Receptors Mediate “Missing Self” Recognition In Vivo

Although it is well established that human NK cells are able to detect the absence of autologous HLA class I in vitro by virtue of inhibitory killer Ig-like receptors (KIR), direct evidence that KIR can mediate "missing self" recognition in vivo is lacking. To test this, we generated mice transgenic for a human KIR B-haplotype and HLA-Cw3 on a C57BL/6 background. NK cells in these mice expressed multiple KIR in a stochastic manner, including the HLA-Cw3-specific inhibitory receptor KIR2DL2. KIR and HLA transgenic mice rejected wild-type C57BL/6 spleen cells upon i.v. injection. This rejection was dependent on the presence of the KIR transgene in the host and on the absence of HLA-Cw3 from the injected target cells. Hence, the KIR transgene mediated "missing self" recognition in vivo. We anticipate that this KIR and HLA transgenic mouse will help shed light on KIR and HLA effects in disease and transplantation.

Beyond Stressed Self: Evidence for NKG2D Ligand Expression on Healthy Cells.

The activity of cytotoxic lymphocytes is regulated by the opposing function of stimulatory and inhibitory cell surface receptors. According to the now classical model of Natural Killer (NK) cell activity, the ligands for inhibitory receptors are constitutively expressed on healthy cells but can be lost on infection and on malignant cells. Loss of inhibitory checks will then allow activating signals to predominate, forming the basis of 'missing self recognition'. Natural Killer Group 2D (NKG2D) is an important member of the cohort of activating receptors expressed on Natural Killer (NK) cells and subsets of T cells. Ligands for the NKG2D receptor comprise a diverse array of self-proteins structurally related to MHC class I molecules. Expression of NKG2D ligands can be induced in cells during infection with pathogens, tumourigenesis, and by stimuli such as DNA damage, oxidative stress, and heat shock. Consequently NKG2D has been widely described as participating in 'stressed self' or 'damaged self' recognition. However, a body of evidence has recently emerged to suggest that this intuitive model of NKG2D function may be an oversimplification. NKG2D ligand expression has now widely been reported on cells that could not be described as stressed or damaged. For example activated T cells can express NKG2D ligands, and constitutive expression of NKG2D ligands has been reported on normal myelomonocytic cells, dendritic cells, and epithelial cells of the gut mucosa. In this article we will review the literature suggesting that NKG2D may function to recognise non-stressed cells and discuss the role NKG2D ligands could be playing in apparently healthy cells.

Cutting Edge: Viral Infection Breaks NK Cell Tolerance to “Missing Self”

NK cells attack cells lacking MHC class I, yet MHC class I-deficient mice have normal numbers of NK cells with intact, albeit diminished, functions. Moreover, wild-type NK cells are tolerant of MHC class I-deficient cells in mixed bone marrow chimeras. In this study, we investigated how the absence of MHC class I affects NK cells. NK cells from beta(2)-microglobulin-deficient (B2m(-/-)) and wild-type mice exhibit similar phenotypic and functional characteristics. Both B2m(-/-) and wild-type Ly49H(+) NK cells proliferated robustly and produced IFN-gamma after infection with mouse CMV. NK cells in mixed wild-type:B2m(-/-) chimeric mice were initially tolerant of MHC class I-deficient host cells. However, this tolerance was gradually lost over time and after mouse CMV infection was rapidly broken, with a pronounced rejection of host B2m(-/-) hematopoietic cells. Thus, although NK cells can be held in check against "missing self," acute inflammation driven by infection can rapidly break established self-tolerance.

Greater HLA Disparity Is Associated with Reduced Risk of Relapse and Improved Event-Free Survival after Nonmyeloablative, HLA-Haploidentical BMT with Post-Transplantation High-Dose Cyclophosphamide

Background: Although a lower relapse risk has been reported after allogeneic BMT with increasing HLA disparity, this potential benefit has been offset by higher rates of acute graft-versus-host disease (GVHD) and non-relapse mortality (NRM). However, it is possible that the type of GVHD prophylaxis could influence the balance between GVHD toxicity and relapse.Patients and methods: We retrospectively analyzed the outcomes of 185 patients with poor-risk hematologic malignancies enrolled on three similar clinical trials of related-donor, haploidentical BMT incorporating high-dose post-transplantation cyclophosphamide (Cy) for GVHD and graft rejection prophylaxis (as published in BBMT 2008; 14:641–50). Molecular typing was at an allele level for HLA-A, -B, -Cw, and -DRB1 and at an allele group level for -DQB1. All received Cy (14.5 mg/kg IV on days −6, −5), fludarabine (30 mg/m2 IV on days −6 to −2), total body irradiation (200 cGy on day −1), and non-T-cell depleted bone marrow infusion. GVHD prophylaxis consisted of Cy (50 mg/kg IV) either once (on day 3; n = 48) or twice (on days 3, 4; n = 137), mycophenolate mofetil for 35 days, and tacrolimus for up to 6 months, with filgrastim begun after the last dose of Cy. Most patients (median age 50, range 1–71) had advanced disease and 49 (26%) had failed autologous BMT. Diagnoses were MDS (22), CMML (3), acute leukemia or lymphoblastic lymphoma (58), CML (11), CLL (15), multiple myeloma (9), non-Hodgkin lymphoma (42), and Hodgkin lymphoma (25).Results: Median follow-up after BMT is 20 months (range, 2–71 months) in those without events. Nonengraftment attributed to primary graft failure or to residual bone marrow malignancy occurred in 29 of 177 evaluable patients (16%). Cumulative incidences of grade II–IV acute GVHD and chronic GVHD were 31% and 15%, respectively. Cumulative incidences of NRM and relapse or progression at one year were 15% and 50%, respectively. Actuarial event-free survival (EFS) at one year was 35%, with grade II–IV acute GVHD by day 100 associated with a trend toward lower cumulative incidence of relapse (p = 0.08) but a significantly higher cumulative incidence of NRM (p = 0.002) on subgroup analysis. Notably, increasing degrees of HLA mismatch at either class I or class II loci had no significant effect on cumulative incidence of acute or chronic GVHD or NRM. In contrast, the presence of a DRB1 antigen mismatch in the GVH, but not host-versus-graft (HVG), direction was associated with a significantly lower cumulative incidence of relapse (Figure a; p = 0.04) and improved EFS (Figure c; p = 0.009), whereas DQB1 antigen and class II allele mismatch status had no effect. Additionally, the presence of two or more class I allele mismatches (composite of A, B, and Cw) in either direction was associated with a significantly lower cumulative incidence of relapse (Figure b; p = 0.045 for GVH direction, p = 0.01 for HVG direction) and improved EFS (Figure d; p = 0.07 for GVH direction, p = 0.001 for HVG direction).Conclusion: Greater HLA disparity appears to be beneficial after nonmyeloablative, HLA-haploidentical BMT that incorporates high-dose post-transplantation Cy. These results suggest an anti-tumor effect of partially HLA-mismatched BMT that is irrespective of clinically significant GVHD. Potential effectors of anti-tumor immunity include HLA-DRB1 reactive CD4+ T-cells, class I reactive CD8+ T-cells, and/or natural killer cells recognizing missing self. Since most patients have several potential HLA-haploidentical related donors, the results support a strategy of choosing a donor who is incompatible for both HLA-DRB1 antigen and multiple HLA class I alleles. [Display omitted]

Self‐tolerance of Human Natural Killer Cells Lacking Self‐HLA‐specific Inhibitory Receptors

Natural killer (NK) cells identify cells with altered human leucocyte antigen (HLA) expression as targets through lacking engagement of self-HLA-specific inhibitory receptors (e.g. killer cell immunoglobulin-like receptor, KIR). Thus, they eliminate cells with 'missing self' because of viral or malignant transformation. We performed analysis of HLA, KIR genotypes and KIR receptor expression patterns at single cell level in NK cells in 17 donors. The function of NK cell subsets is determined by degranulation assays using target cells expressing self, cognate, control or no HLA class I. Donors could be grouped into three groups: their NK cells possess potential for alloreactivity, autoreactivity based on the presence of NK cells expressing particular KIR only (mono-KIR) in the absence of its ligand or lack alloreactivity. All donors possess NK cells lacking all detectable inhibitory receptors. Both potential autoreactive subpopulations did not respond to HLA class I-positive target cells. They retain partial reactivity against HLA class I-negative tumour target cells. Mono-KIR NK cells without the corresponding ligands in the individuals and NK cells lacking all inhibitory receptors behave self-tolerant. Our results suggest alternative mechanisms than HLA-specific inhibitory receptors to control NK cell activity. But HLA seems to be involved in shaping effector function of the NK cell repertoire.

The Fetal Allograft Revisited: Does the Study of an Ancient Invertebrate Species Shed Light on the Role of Natural Killer Cells at the Maternal-Fetal Interface?

Human pregnancy poses a fundamental immunological problem because the placenta and fetus are genetically different from the host mother. Classical transplantation theory has not provided a plausible solution to this problem. Study of naturally occurring allogeneic chimeras in the colonial marine invertebrate, Botryllus schlosseri, has yielded fresh insight into the primitive development of allorecognition, especially regarding the role of natural killer (NK) cells. Uterine NK cells have a unique phenotype that appears to parallel aspects of the NK-like cells in the allorecognition system of B. schlosseri. Most notably, both cell types recognize and reject “missing self” and both are involved in the generation of a common vascular system between two individuals. Chimeric combination in B. schlosseri results in vascular fusion between two individual colonies; uterine NK cells appear essential to the establishment of adequate maternal-fetal circulation. Since human uterine NK cells appear to de-emphasize primary immunological function, it is proposed that they may share the same evolutionary roots as the B. schlosseri allorecognition system rather than a primary origin in immunity.

A novel immunoglobulin-like transcript from rainbow trout with two Ig-like domains and two isoforms

Within the innate immune response in primates the nonrearranging killer immunoglobulin-like receptors (KIR) enable natural killer cells to discern target cells exposing “missing self” signals. Recently the novel immune-type receptor (NITR) and the novel immunoglobulin-like transcript (NILT) gene families have been discovered in fish encoding nonrearranging receptors with a similar molecular structure to that of KIRs. Besides the structural similarity the high degree of haplotypic and allelic variation suggests these genes to be functional KIR homologs and involved in recognizing self-determinants in lower vertebrates. Whereas numerous NITR sequences have been detected in several fish species only two NILT genes have been published for carp yet. Here we report a first rainbow trout NILT sequence, Onmy-NILT2D, alternatively spliced into a long membrane-bound and a short, putatively secreted form, both with the same two immunoglobulin (Ig)-like domains. The second Ig-like domain comprises a consensus pattern present both in NILTs and NITRs. The cytoplasmic region of the long form simultaneously contains immunoreceptor tyrosine-based inhibitory motifs (ITIM) and an immunoreceptor tyrosine-based activating motif (ITAM).

The Activating KIR2DS2 Gene Influences NK Alloreactivity and NK Repertoire.

The hematopoietic stem cell transplant (HCT) donor KIR genotype has been correlated with disease-free survival in patients with acute myelogenous leukemia. The Killer Cell Immunoglobulin-like Receptor (KIR) gene family encodes highly homologous pairs of activating and inhibiting receptors, 2DL1–2DS1; 2DL2/3–2DS2; and 3DL1–3DS1. Inhibitory members are known to regulate NK cell function through interactions with HLA Class I antigens. The role of activating KIRs and their ligand specificity is, however, not well defined. The activating receptor, KIR2DS1, is known to bind the HLA-Cw C2 group antigens and we have recently demonstrated a role for this receptor in NK cell allorecognition. In contrast, KIR2DS2 does not bind HLA-Cw C1 group antigens, and a functional role of this receptor even in NK allorecognition has not been established. We now demonstrate, that presence of the activating KIR2DS2 gene in NK donors homozygous for the HLA-KIR ligand group C2 is associated with significant alloreactivity against C1 homozygous target cells (polyclonal NK cells, p=0.006; NK clones, p=0.001). This alloreactivity is mediated by “missing self” on the target and is dominated by “lack of C2 group on target”. The “missing C2” effect was absent, however, in C2 homozygous donors lacking 2DS2 (p=0.99). Only very rare cytotoxic NK clones expressing GL183 (2DL2/3, 2DS2) and with alloreactivity against C1 targets could be generated in vitro from 2DS2-positive, C2 homozygous donors. A majority of these rare GL183-positive clones did not demonstrate inhibitory function against the HLA class I deficient 721.221 transfected with Cw3 (C1-group), and GL183 cross-linking of the clones resulted in increased cytokine production. Thus, KIR2DS2 is an activating receptor in NK clones from C2 homozygous donors, but does not appear to recognize C1 ligand. We next investigated 2DS2 function in donors heterozygous for the C groups (i.e. C1/C2). Analysis of NK cell function in a 2DS2-positive, C1/C2 donor revealed a “missing HLA-KIR ligand” effect for the C2 group. Cytotoxicity by IL2-propagated, polyclonal NK cells and NK clones revealed allocytotoxicity against targets lacking the C2 group (p<0.001). In addition, a repertoire analysis on 138 NK clones generated from this donor revealed a marked increase in the number of EB6 (KIR2DL1/S1)-expressing NK clones (95%) compared to both the fresh (10%–50%) and the IL-2-expanded polyclonal NK repertoire (12%–60%). Additionally, all EB6-expressing clones from this donor were inhibited by the C2 ligand. Subsequent studies in freshly isolated NK cells following activating receptor cross-linking (NKp46, NKG2C, and CD16) or by alloantigen activation demonstrated that the functioning subset of NK cells in this donor predominantly expressed the EB6 receptor. Other inhibitory receptors (e.g. NKG2A, KIR3DL1, and KIR3DL2) did not contribute significantly to the functional subset of NK cells. Presence of 2DS2 in this donor was therefore associated with a “skewing” of the NK repertoire towards EB6 positivity, and dominated by functional NK cells that were inhibited by the “self” C2 ligand. Collectively, these studies provide the first evidence that activating KIR can influence the NK cell repertoire. Furthermore, our studies would indicate that presence of activating KIRs in HCT donors for recipients homozygous HLA-KIR ligands might induce post-transplantation graft versus host NK alloreactivity.

KIR2DS1-Positive NK Cells Mediate Alloresponse against the C2 HLA-KIR Ligand Group In Vitro

The inhibitory 2DL1 and activating 2DS1 killer Ig-like receptors (KIR) both have shared ligand specificity for codon sequences in the C2 group HLA-Cw Ags. In this study, we have investigated NK cell activation by allogeneic target cells expressing different combinations of the HLA-KIR ligand groups C1, C2, and Bw4. We demonstrate that fresh NK cells as well as IL-2-propagated NK cells from 2DS1-positive donors that are homozygous for the C1 ligand group are activated in vitro by B lymphoblastoid cell lines expressing the C2 group. This response is, in part, due to the absence of C1 group recognition mediated by the inhibitory receptor 2DL2/3. This "missing self" alloresponse to C2, however, is rarely observed in NK cells from donors lacking 2DS1. Even in presence of 2DS1, the NK alloresponse is dramatically reduced in donors that have C2 group as "self." Analysis of selected NK clones that express 2DS1 mRNA and lack mRNA for 2DL1 demonstrates that activation by the C2 ligand and mAb cross-linking of 2DS1 in these clones induces IFN-gamma. Furthermore, this C2 group-induced activation is inhibited by Abs to both HLA class I and the receptor. Collectively, these studies demonstrate that NK cells from 2DS1-positive donors are activated by target cells that express the C2 group as an alloantigen. This leads to increased IFN-gamma-positive fresh NK cells and induces NK allocytotoxicity in IL2-propagated polyclonal NK cells and NK clones. This study also provides support for the concept that incompatibility for the HLA-KIR ligand groups C1, C2, and Bw4 dominates NK alloactivation in vitro.

Natural killer cell alloreactivity in allogeneic hematopoietic transplantation

This review will focus on the translation of natural killer cell recognition of missing self into the clinical practice of allogeneic hematopoietic transplantation and discuss how it has opened innovative perspectives in the cure of leukemia. Allogeneic hematopoietic stem cell transplantation from a human leukocyte antigen-matched sibling can cure leukemia but 75% of patients do not have a matched donor, one alternative source of stem cells includes full haplotype mismatched family members. As haploidentical transplantation must be extensively T cell depleted to prevent lethal graft-versus-host disease, it cannot rely on donor T cells for the graft-versus-leukemia effect. Mismatched transplantation, however, triggers alloreactivity mediated by natural killer cells which is based upon 'missing self recognition'. Recent studies using preclinical murine models of haploidentical transplantation demonstrated that conditioning with alloreactive natural killer cells ablates the recipient immune system and leukemia cells. In the clinical setting of mismatched hematopoietic stem cell transplantation, donor versus recipient natural killer cell alloreactivity has been associated with better outcome, particularly in patients with acute myeloid leukemia who are transplanted in remission. Given the benefits of natural killer cell alloreactivity, it is expected that it will encourage greater use of haploidentical transplants for the large numbers of leukemia patients without matched donors.

226: Differential requirement for NKG2D in the rejection of haplomismatched and MHC-I deficient bone marrow

Mouse Natural Killer (NK) cells mediate rejection of MHC-mismatched or MHC-deficient bone marrow allografts. Counteracting activating and inhibitory signals regulate NK cell activity. The ability of F1 mice to reject parental bone marrow (BM) cells (hybrid resistance) has been attributed to the presence in the recipient of a subset of NK cells that lack inhibitory receptors for the MHC class I antigens expressed by the donor cells. Evidence supporting the “missing self” hypothesis of hybrid resistance was provided by demonstration that MHC class-I deficient donor BM is rejected by otherwise syngeneic recipients. We have previously demonstrated a role for NKG2D, an activating NK cell receptor, in rejection of Balb/c (H-2d) parental BM by (Balb/c x C57Bl/6; H-2b/d) F1 (CB6F1) recipient mice. NKG2D ligands are expressed on the regenerating myeloid BM cells in Balb/c and NOD mice but not substantially on the regenerating C57Bl/6 BM cells. Moreover, the rejection of Balb/c BM by CB6F1 recipients was blocked by a neutralizing anti-NKG2D monoclonal antibody. The purpose of this study was to determine whether NKG2D blockade was sufficient to prevent rejection of MHC-I deficient BM. Beta-2 microglobulin deficient (B2M-/-) Balb/c or NOD bone marrow was transplanted into irradiated CB6F1 mice that had been pretreated with neutralizing antibodies to NKG2D. In contrast to results with wild-type Balb/c or NOD BM whose rejection was prevented by anti-NKG2D, B2M-/- donor cells were rejected despite NKG2D blockade. Furthermore, syngeneic MHC class I+ Balb/c recipients were able to reject B2M-/- Balb/c donors despite NKG2D blockade. These results suggest that rejection of MHC class I-bearing BM requires activation via NKG2D, whereas MHC class I-deficient BM elicit a sufficiently strong NK response that augmentation by NKG2D signaling is not essential for the rejection. Therefore, the hybrid resistance model in which MHC class I-bearing BM are used for transplantation may better reflect the situation in human hematopoietic stem cell transplantation. Mouse Natural Killer (NK) cells mediate rejection of MHC-mismatched or MHC-deficient bone marrow allografts. Counteracting activating and inhibitory signals regulate NK cell activity. The ability of F1 mice to reject parental bone marrow (BM) cells (hybrid resistance) has been attributed to the presence in the recipient of a subset of NK cells that lack inhibitory receptors for the MHC class I antigens expressed by the donor cells. Evidence supporting the “missing self” hypothesis of hybrid resistance was provided by demonstration that MHC class-I deficient donor BM is rejected by otherwise syngeneic recipients. We have previously demonstrated a role for NKG2D, an activating NK cell receptor, in rejection of Balb/c (H-2d) parental BM by (Balb/c x C57Bl/6; H-2b/d) F1 (CB6F1) recipient mice. NKG2D ligands are expressed on the regenerating myeloid BM cells in Balb/c and NOD mice but not substantially on the regenerating C57Bl/6 BM cells. Moreover, the rejection of Balb/c BM by CB6F1 recipients was blocked by a neutralizing anti-NKG2D monoclonal antibody. The purpose of this study was to determine whether NKG2D blockade was sufficient to prevent rejection of MHC-I deficient BM. Beta-2 microglobulin deficient (B2M-/-) Balb/c or NOD bone marrow was transplanted into irradiated CB6F1 mice that had been pretreated with neutralizing antibodies to NKG2D. In contrast to results with wild-type Balb/c or NOD BM whose rejection was prevented by anti-NKG2D, B2M-/- donor cells were rejected despite NKG2D blockade. Furthermore, syngeneic MHC class I+ Balb/c recipients were able to reject B2M-/- Balb/c donors despite NKG2D blockade. These results suggest that rejection of MHC class I-bearing BM requires activation via NKG2D, whereas MHC class I-deficient BM elicit a sufficiently strong NK response that augmentation by NKG2D signaling is not essential for the rejection. Therefore, the hybrid resistance model in which MHC class I-bearing BM are used for transplantation may better reflect the situation in human hematopoietic stem cell transplantation.

Donor Natural Killer Cell Allorecognition of Missing Self in Haploidentical Hematopoietic Transplantation for Acute Myeloid Leukemia: Challenging Its Predictive Value.

In haploidentical transplants that are KIR ligand mismatched in the GvH direction, functional donor NK-cells that express as their sole inhibitory receptor for self, a KIR for the HLA-class-I group which is absent in the recipient, sense the missing expression of the self class-I ligand on allogeneic targets and mediate alloreactions. In a limited series of transplants donor-vs-recipient NK-cell alloreactivity reduced the risk of AML relapse and markedly improved EFS (Ruggeri L, Science 2002). Here, we analyzed 112 AML patients transplanted from NK-alloreactive (n=51) or non-NK-alloreactive (n=61) haploidentical donors. NK-alloreactive donors possessed: HLA-class-I KIR ligand(s) which were missing in the recipients, KIR gene(s) for missing self recognition on recipient targets, and alloreactive NK-clones against recipient targets. Transplantation from NK-alloreactive donors was associated with: significantly lower relapse rate in patients transplanted in CR (3% vs 47%) (P<0.003); decrease infectious mortality which was more evident in patients transplanted in relapse (P=0.1); better EFS in patients transplanted in relapse (34% vs 6%, P=0.04) and in remission (67% vs 18%, P=0.02); reduced risk of relapse or death (relative risk vs non-NK-alloreactive donor: 0.48 [95% CI 0.29–0.78], P<0.001). Recently, an additional algorithm, termed the “missing ligand” model, has been proposed for predicting favorable outcomes not only in haploidentical (Leung W, JI 2004; JI 2005) but also in matched sibling (Hsu KC, Blood 2005) and in unrelated donor transplants (Hsu KC, BBMT 2006). The “missing-ligand” model includes all donor-recipient pairs in whom there is a mismatch between KIR(s) in the donor and HLA molecule(s) in the recipient. Consequently, the model includes all KIR ligand-mismatched transplants because they are all associated with a missing KIR ligand in the recipient, and KIR ligand-matched transplants from donors possessing “extra” KIR(s) for which neither donor nor recipient have HLA ligand(s). Therefore, in the same series of patients we tested the “missing ligand” model. The first step was to divide our 61 non-NK alloreactive (KIR ligand-matched) donor-recipient pairs according to the number of KIR ligands in donor and recipient, i.e., three KIR ligands (29 patients; 15 in remission, 14 in relapse at transplant) vs fewer than three (32 patients; 16 in remission, 16 in relapse at transplant). EFS did not differ in each sub-group. Both curves indicated worse survival than after transplantation from NK-alloreactive donors. The second step was to group the above 32 “missing ligand” transplants and all 51 KIR ligand-mismatched transplants (which corresponded to all our NK-alloreactive transplants). We analyzed EFS in this pool of 83 patients (46 in remission, 37 in relapse at transplant) against EFS in 29 patients with no missing ligand (15 in remission, 14 in relapse at transplant). No significant difference emerged. EFS in the “missing ligand” cohort was worse than after transplantation from NK-alloreactive donors. Therefore, the present analysis leaves no doubt that KIR ligand mismatches, i.e., donor NK cell recognition of “missing self” on recipient targets, are essential for triggering powerful NK cell alloreactions that impact beneficially on transplantation outcomes.

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

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.

The First Molecular Basis of the “Missing Self” Hypothesis

I still remember the day my advisor, Dr. Rafael Solana, came to me with the article of Karlhofer et al. ([1][1]) in his hands and told me that that paper was the proof for the receptor inhibition model of the “missing self” hypothesis. I had just joined the Laboratory of Immunology in the

Lymphocyte activation in response to melanoma: interaction of NK-associated receptors and their ligands.

In recent years, studies on the molecular and cellular mechanisms of immune responses against melanoma have contributed to a better understanding of how these tumours can be recognised by cytotoxic cells and the mechanisms they have developed to escape from innate and adaptive immunity. Lysis of melanoma cells by natural killer (NK) cells and cytolytic T cells is the result of a fine balance between signals transmitted by activating and inhibitory receptors. In addition to the T cell receptor, these were initially described as NK cell-associated receptors (NKRs) and were later also found on subsets of T lymphocytes, particularly effector-memory and terminally differentiated CD8 T cells. An increase of NKR(+)CD8(+) T cells has been found in melanoma patients, correlating with the expansion of differentiated effector CD8(+)CD28(null) CD27(null) T cells. NKRs can regulate the lysis of target cells expressing appropriate ligands. Activating receptors recognise ligands on tumours whereas inhibitory receptors are specific for MHC class I antigens and sense missing self. Altered expression of MHC class I antigens is frequently found on melanoma cells, preventing recognition by specific cytolytic T cells but favouring NK cell recognition. Changes in the expression of NKR-ligands in melanoma contribute in explaining the differences in the capacity of cytotoxic immune cells to control melanoma growth and dissemination.