Natural killer cell education in mice with single or multiple major histocompatibility complex class I molecules

The ability of murine NK cells to reject cells lacking self MHC class I expression results from an in vivo education process. To study the impact of individual MHC class I alleles on this process, we generated mice expressing single MHC class I alleles (K(b), D(b), D(d), or L(d)) or combinations of two or more alleles. All single MHC class I mice rejected MHC class I-deficient cells in an NK cell-dependent way. Expression of K(b) or D(d) conveyed strong rejection of MHC class I-deficient cells, whereas the expression of D(b) or L(d) resulted in weaker responses. The educating impact of weak ligands (D(b) and L(d)) was further attenuated by the introduction of additional MHC class I alleles, whereas strong ligands (K(b) and D(d)) maintained their educating impact under such conditions. An analysis of activating and inhibitory receptors in single MHC class I mice suggested that the educating impact of a given MHC class I molecule was controlled both by the number of NK cells affected and by the strength of each MHC class I-Ly49 receptor interaction, indicating that NK cell education may be regulated by a combination of qualitative and quantitative events.     

Regulatory mechanisms of NK cell functions

Natural Killer (NK) cells discriminate self from non-self in a manner distinct from T cells. NK cells exhibit cytotoxicity against "missing-self" by killing any cells in principle except normal self-cells. Cells expressing low levels of self MHC class I molecules such as tumor cells and foreign cells are killed, whereas normal self cells are neglected by NK cells. Although identities of activation receptors triggering NK activity are still unclear, recent studies have revealed molecules inhibiting cytotoxicity against normal self cells. In this review, we summarize current understanding of molecular basis for missing-self hypothesis and control mechanisms of NK cell activation.     

Cytomegalovirus immunoevasin reveals the physiological role of "missing self" recognition in natural killer cell dependent virus control in vivo

Cytomegaloviruses (CMVs) are renowned for interfering with the immune system of their hosts. To sidestep antigen presentation and destruction by CD8(+) T cells, these viruses reduce expression of major histocompatibility complex class I (MHC I) molecules. However, this process sensitizes the virus-infected cells to natural killer (NK) cell-mediated killing via the "missing self" axis. Mouse cytomegalovirus (MCMV) uses m152 and m06 encoded proteins to inhibit surface expression of MHC I molecules. In addition, it encodes another protein, m04, which forms complexes with MHC I and escorts them to the cell surface. This mechanism is believed to prevent NK cell activation and killing by restoring the "self" signature and allowing the engagement of inhibitory Ly49 receptors on NK cells. Here we show that MCMV lacking m04 was attenuated in an NK cell- and MHC I-dependent manner. NK cell-mediated control of the infection was dependent on the presence of NK cell subsets expressing different inhibitory Ly49 receptors. In addition to providing evidence for immunoevasion strategies used by CMVs to avoid NK cell control via the missing-self pathway, our study is the first to demonstrate that missing self-dependent NK cell activation is biologically relevant in the protection against viral infection in vivo.     

Involvement of HLA class I alleles in natural killer (NK) cell-specific functions: expression of HLA-Cw3 confers selective protection from lysis by alloreactive NK clones displaying a defined specificity (specificity 2).

This study was designed to identify the target molecules of the natural killer (NK) cell-mediated recognition of normal allogeneic target cells. As previously shown, the gene(s) governing the first NK-defined allospecificity (specificity 1) were found to be localized in the major histocompatibility complex region between BF gene and HLA-A. In addition, the analysis of a previously described family revealed that a donor (donor 81) was heterozygous for three distinct NK-defined allospecificities (specificities 1, 2, and 5). HLA variants were derived from the B-Epstein-Barr virus cell line of donor 81 by gamma irradiation followed by negative selection using monoclonal antibodies specific for the appropriate HLA allele. Several variants were derived that lacked one or more class I antigen expressions. These variants were analyzed for the susceptibility to lysis by NK clones recognizing different allospecificities. The loss of HLA-A did not modify the phenotype (i.e., "resistance to lysis"). On the other hand, a variant lacking expression of all class I antigens became susceptible to lysis by all alloreactive clones. Variants characterized by the selective loss of class I antigens coded for by the maternal chromosome became susceptible to lysis by anti-2-specific clones. Conversely, variants selectively lacking class I antigens coded for by paternal chromosome became susceptible to lysis by anti-1 and anti-5 clones (but not by anti-2 clones). Since the Cw3 allele was lost in the variant that acquired susceptibility to lysis by anti-2 clones and, in informative families, it was found to cosegregate with the character "resistance to lysis" by anti-2 clones, we analyzed whether Cw3 could represent the element conferring selective resistance to lysis by anti-2 clones. To this end, murine P815 cells transfected with HLA Cw3 (or with other HLA class I genes) were used as target cells in a cytolytic assay in which effector cells were represented by alloreactive NK clones directed against different specificities. Anti-2-specific clones efficiently lysed untransfected or A2-, A3-, and A24-transfected P815 cells, while they failed to lyse Cw3-transfected cells. NK clones recognizing specificities other than specificity 2 lysed untransfected or Cw3-transfected cells. Thus, the loss of Cw3 resulted in the de novo appearance of susceptibility to lysis, and transfection of the HLA-negative P815 cells with Cw3 resulted in resistance to lysis by anti-2 clones. Therefore, we can infer that Cw3 expression on (both human and murine) target cells confers selective protection from lysis mediated by anti-2 NK clones.     

Acquisition of external major histocompatibility complex class I molecules by natural killer cells expressing inhibitory Ly49 receptors.

Murine natural killer (NK) cells express inhibitory Ly49 receptors specific for major histocompatibility complex (MHC) class I molecules. We report that during interactions with cells in the environment, NK cells acquired MHC class I ligands from surrounding cells in a Ly49-specific fashion and displayed them at the cell surface. Ligand acquisition sometimes reached 20% of the MHC class I expression on surrounding cells, involved transfer of the entire MHC class I protein to the NK cell, and was independent of whether or not the NK cell expressed the MHC class I ligand itself. We also present indirect evidence for spontaneous MHC class I acquisition in vivo, as well as describe an in vitro coculture system with transfected cells in which the same phenomenon occurred. Functional studies in the latter model showed that uptake of H-2D(d) by Ly49A+ NK cells was accompanied by a partial inactivation of cytotoxic activity in the NK cell, as tested against H-2D(d)-negative target cells. In addition, ligand acquisition did not abrogate the ability of Ly49A+ NK cells to receive inhibitory signals from external H-2D(d) molecules. This study is the first to describe ligand acquisition by NK cells, which parallels recently described phenomena in T and B cells.     

Split tolerance induced by the intrathymic adoptive transfer of thymocyte stem cells

The intrathymic transfer of semiallogeneic CD4/CD8 double-negative (DN) thymocyte stem cells into irradiated host mice resulted in a transient state of chimerism in adoptive host thymus, spleen, and lymph nodes. Host-derived T cells, isolated from the thymus and periphery of the chimeric mice, were found to be specifically nonresponsive to the MHC antigens of the semiallogeneic DN donor in cytotoxicity assays. This nonresponsiveness was not permanent, but persisted as long as appreciable numbers of Thy-1 alloantigen-positive progeny of the DN donor cells could be detected in the spleen and lymph nodes of adoptive host mice. FACS sorting of DN donor cells before intrathymic transfer indicated that nonresponsiveness could be induced by Thy-1+ cells and was therefore not attributable to contaminating thymic macrophages, dendritic cells, or B cells. When FACS-sorted Thy-1+ (bm5 x bm12)F1 DN cells were transferred intrathymically into C57BL/6 hosts, nonresponsiveness to DN donor MHC class I but not class II alloantigen (split tolerance) was observed. These experiments were repeated using FACS-sorted Thy-1+ DN donor cells that were semiallogeneic to the irradiated adoptive host at either MHC class I or class II locus with similar results. Limiting dilution analysis showed that host-derived CTL precursors were tolerant of DN donor MHC class I alloantigen and no evidence for the involvement of suppressor T cells was found. The data indicate that murine thymocytes themselves are capable of tolerizing to MHC class I but not class II alloantigen after intrathymic transfer. The implications for intrathymic T cell differentiation and maintenance of self tolerance are discussed.     

MHC class I receptors on natural killer cells: on with the old and in with the new

The functions of natural killer (NK) cells are controlled by an abundance of activating and inhibitory receptors. Many of these interact with MHC class I molecules. The MHC system also interacts with cytotoxic T-lymphocytes and has been shown to comprise a rapidly evolving family of molecules. This challenges the functional relationship of NK cell receptors with their ligands. Although individual receptors have become subject to species-specific expansions over evolutionary time, the main themes of the NK cell interaction with MHC class I have been preserved. This review details the interaction of NK cell receptors with MHC class I and discusses their unexpectedly rapid evolution.     

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

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.NK cells are innate immune lymphocytes with potent effector functions against infected and tumor cells. NK cells integrate signals received through target cell ligand-mediated engagement of activation receptors with those from inhibitory receptor engagement by MHC class I ligands expressed on targets. Absence of MHC class I on target cells often leads to NK cell activation. This phenomenon, termed “missing-self,” allows NK cells to attack and eliminate cells with aberrantly low or absent expression of MHC class I (Kärre et al., 1986), as with transformed or virally infected cells. Hence, NK cells use inhibitory receptors to assess the surface of self-tissues for MHC class I expression, providing a line of defense against pathogens and abnormal cell growth.MHC class I molecules are also crucial to acquisition of effector function by NK cells in vivo as NK cells from MHC class I–deficient hosts are defective in natural killing (Bix et al., 1991; Höglund et al., 1991) and hyporesponsive to triggering through their activation receptors (Fernandez et al., 2005; Kim et al., 2005). Recent data obtained in MHC-sufficient hosts support the hypothesis that cognate interaction between inhibitory receptors and self-MHC is necessary for acquisition of effector function. For example, Ly49C⁺ NK cells, which bind a self-MHC I ligand (H2K^b) in the H2^b haplotype of C57BL/6 mice, display more robust production of cytokines upon stimulation than NK cells expressing only Ly49A, which has no H2^b ligand (Kim et al., 2005). This is most evident in a C57BL/6 transgenic (Tg) mouse expressing a single-chain trimer H2K^b (SCT-K^b) molecule, consisting of the H2K^b heavy chain covalently linked to β₂-microglobulin (β₂m) and the SIINFEKL peptide from ovalbumin. In mice where SCT-K^b is the only MHC class I molecule expressed, i.e., SCT-K^b Tg mouse on the K^b−/−D^b−/−β₂m^−/− (triple KO; TKO) background, and Ly49C is the sole NK cell receptor capable of binding the SCT-K^b molecule, only Ly49C⁺ NK cells display the licensed phenotype (Kim et al., 2005). Thus, engagement of self–MHC-specific receptor “licenses” NK cells to be functionally competent to be triggered through their activation receptors.Although there has been debate on the meaning of the term “licensing,” most groups now agree that the engagement of inhibitory receptors by self–MHC class I results in education of NK cells to become functionally competent (Anfossi et al., 2006; Raulet and Vance, 2006). Moreover, such education effects have also been observed with human NK cells via self-HLA recognition by killer immunoglobulin-like receptors (KIRs), which are related by convergent evolution to murine Ly49 receptors (Kelley et al., 2005; Anfossi et al., 2006; Yu et al., 2007; Kim et al., 2008). Thus, self–MHC class I engagement by NK cell inhibitory receptors appears to be a fundamental element in acquisition of NK cell effector function.Conventional murine NK cells are thought to develop primarily, if not completely, in the BM where they progress through a series of stages en route to full maturation (Di Santo, 2006; Kim et al., 2002). After this process, they leave the BM and populate the peripheral tissues. Egress from the BM is generally accepted as a marker of mature conventional NK cells, as cells isolated from peripheral lymphoid tissues demonstrate effector function upon stimulation. We previously hypothesized licensing to be a developmental process because Ly49 receptors are first expressed at an immature stage during NK cell maturation in the BM (Kim et al., 2002). Thereafter, developing NK cells undergo constitutive proliferation that appears to be influenced by MHC class I and the relevant Ly49 receptor (Kim et al., 2005). However, it is not known if licensing can occur outside the context of development within the BM, or whether the unlicensed phenotype is fixed.Herein, we performed adoptive transfer of peripheral, hyporesponsive NK cells from MHC class I–deficient donors to MHC class I–sufficient hosts, resulting in the generation of functional donor NK cells. Expression of a host MHC class I ligand specific for a donor cell inhibitory NK cell receptor was necessary for the observed gain of function, which is highly suggestive of licensing as a mechanism. We conclude that the unlicensed phenotype is not fixed in apparently mature peripheral NK cells, and that licensing may not be exclusively a developmental process in the BM.     

H-2-restricted cytolytic T lymphocytes specific for HLA display T cell receptors of limited diversity.

We previously showed that H-2Kd-restricted cytotoxic T lymphocyte (CTL) clones specific for a single nonapeptide derived from the Plasmodium berghei circumsporozoite (PbCS) protein displayed T cell receptors (TCRs) of highly diverse primary structure. We have now analyzed the TCR repertoire of CTLs that recognize a peptide derived from the human class I major histocompatibility complex (MHC) molecule HLA-Cw3 in association with the same murine class I MHC molecule H-2Kd. We first sequenced the TCR alpha and beta genes of the CTL clone Cw3/1.1 and, based on this genomic analysis, the TCR alpha and beta cDNA junctional regions of 23 independent H-2Kd-restricted CTL clones specific for HLA-Cw3. The results show that the TCR chains display very limited heterogeneity, both in terms of V alpha, J alpha, V beta, and J beta segments, and in terms of length and sequence of the CDR3 alpha and beta loops. The TCR repertoire used in vivo was then analyzed by harvesting CTL populations from the peritoneal cavity of immune mice. The peritoneal exudate lymphocytes (PELs) displayed HLA-Cw3-specific cytolytic activity in the absence of any stimulation in vitro. Remarkably, most of these freshly isolated PELs expressed TCRs that shared the same structural features as those from HLA-Cw3-reactive CTL clones. Thus, our results show that a peptide from HLA-Cw3 presented by H-2Kd selects CTLs that bear TCRs of very limited diversity in vivo. When taken together with the high diversity of the TCRs specific for the PbCS peptide, these findings suggest that natural tolerance to self peptides presented by class I MHC molecules may substantially reduce the size of the TCR repertoire of CTLs specific for antigenic peptides homologous to self.     

异基因造血干细胞移植中杀伤细胞抑制性受体(KIR)在GVHD和GVL 发生中的作用

杀伤细胞抑制性受体(KIR)属于免疫球蛋白超家族成员Ⅰ型跨膜蛋白分子，主要表达于人NK细胞和某些T淋巴细胞。它们的配体为HLA Ⅰ类分子。当KIR所识别的MHC配体缺失时，即表现为对靶细胞的杀伤作用(所谓丢失自我“missing-self”机制)。已证实，)NK细胞和T细胞的KIR分子与靶细胞的MHC分子特异性的识别机制参与移植物抗宿主病(GVHD)的发生并且影响移植物抗白血病(GVL)作用。KIR的存在可能是免疫活性细胞不攻击自身组织的主要机制。KIR基因家族及其配体HLA-C分子均具有基因多态性，因此在HLA相合和不全相合的异基因造血干细胞移植中，供受者KIR基因表达的差异在一定程度上影响移植效果。本文主要就KIR如何影响异基因造血干细胞移植后GVHD和GVL进行综述。     

Natural Killer Cell Tolerance in Mice with Mosaic Expression of Major Histocompatibility Complex Class I Transgene

We have studied natural killer (NK) cell tolerance in a major histocompatibility complex (MHC) class I transgenic line, DL6, in which the transgene product was expressed on only a fraction of blood cells. In contrast with transgenic mice expressing the same transgene in all cells, NK cells from mosaic mice failed to reject transgene-negative bone marrow or lymphoma grafts. However, they retained the capability to reject cells with a total missing-self phenotype, i.e., cells lacking also wild-type MHC class I molecules. Tolerance against transgene-negative cells was demonstrated also in vitro, and could be broken if transgene-positive spleen cells of mosaic mice were separated from negative cells before, or after 4 d of culture in interleukin-2. The results provide support for selective NK cell tolerance to one particular missing-self phenotype but not to another. We suggest that this tolerance is determined by NK cell interactions with multiple cells in the environment, and that it is dominantly controlled by the presence of cells lacking a specific MHC class I ligand. Furthermore, the tolerant NK cells could be reactivated in vitro, which suggests that the tolerance occurs without deletion of the potentially autoreactive NK cell subset(s), and that it may be dependent upon the continuous presence of tolerizing cells.NK cells kill tumor cells and virus-infected cells (1–3), regulate hematopoiesis (4), and mediate rejection of MHC mismatched hematopoietic grafts (5, 6). The molecular interactions that take place during NK cell recognition are incompletely understood, but one important factor for NK cell sensitivity is the MHC class I expression of the target. In contrast with T cells, which require MHC class I expression by target cells to initiate lysis, NK cells preferentially kill cells lacking MHC class I expression (7–13). However, NK cell recognition does not depend on complete MHC class I deficiency on the graft. Failure of a target cell to express one specific MHC class I allele may be sufficient to trigger NK cells. This mechanism was suggested as one explanation of hybrid resistance, a phenomenon in which NK cells of F₁ hybrid mice reject parental hematopoietic grafts (reviewed in reference 6). According to the missing-self hypothesis, parental cells would be rejected by F₁ hybrid mice because they fail to express a complete set of host MHC class I alleles (7, 9). Evidence for this hypothesis has been obtained in experiments with MHC class I transgenic mice. Introduction of a D^d transgene in C57BL/6 (B6) mice conveyed NK cell–mediated rejection of nontransgenic, but otherwise syngeneic, grafts (14, 15). Transfection of the D^d gene to the sensitive target led to escape from rejection, suggesting that killing was triggered by missing self (16–18).The results described above suggest that the ability to recognize cells lacking one or several specific MHC class I alleles may represent a general strategy in NK cell function. The identification of MHC class I–specific inhibitory receptors on NK cells, such as the members of the Ly-49 family in the mouse (19–21) and the p58/p70 molecules in human (22–24) have recently given molecular support for this concept. When NK cells carrying these receptors meet target cells expressing the correct MHC class I ligands, lysis is inhibited (19, 21, 25, 26). Furthermore, host MHC class I alleles also influence the expression and function of the inhibitory receptors (27–30). These results emphasize the pivotal role of host MHC class I molecules in the development of NK cell specificity, and raise questions as to how MHC class I molecules educate NK cells and how self-tolerance is secured.In contrast with T and B cells, little is known regarding the mechanisms that induce NK cell tolerance and about the properties of the tolerant NK cells. Attempts to induce tolerance in F₁ mice by inoculating parental cells have been made (31–36), but the interpretations of such experiments have been difficult. First, the recipients were mostly adult mice containing mature NK cells, which may not be ideal for the study of how tolerance would develop normally. Second, the inoculated parental cells were in many cases mature immunocompetent cells, which makes it difficult to distinguish between specific tolerizing effects on NK cells and nonspecific effects of graft versus host disease. Third, there have been no studies of NK cell tolerance to cells lacking specific self-MHC class I alleles.In the present report, we have studied the development of the NK cell repertoire and NK cell tolerance to self in an MHC class I transgenic mouse (DL6) in which the transgene D^d/L^d (α1/α2 domains of D^d coupled to the α3 domain, transmembrane and intracellular domains of L^d) is spontaneously expressed in only a fraction (10–80%) of the hematopoietic cells. This model has allowed us to ask a number of questions about the role of host MHC class I molecules in NK cell development. (a) Are the MHC class I molecules expressed by the NK cells themselves sufficient to determine their specificity, or are interactions with other cells necessary? (b) If interactions with other cells are important, would the presence of cells selectively deficient in a particular MHC class I ligand dominantly control tolerance to these cells? Alternatively, would the interaction with cells expressing a particular ligand be sufficient to instruct NK cells to kill cells lacking this ligand? (c) Is tolerance to different missing-self phenotypes controlled selectively and independently? (d) Are potentially autoreactive NK cells deleted in a selection process or can they persist as anergized or specifically tolerized cells? (e) In the latter case, is the specificity of an NK cell a permanent property or can it be altered?     

Antigen recognition by MHC-incompatible cells of a human mismatched chimera

Tetanus toxin (TT)-specific T cell clones of donor origin were obtained from a patient with severe combined immunodeficiency (SCID) successfully reconstituted by transplantation of allogeneic fetal liver and thymus cells from two different donors performed 10 yr ago. A series of these clones recognized TT in the context of "allo" class II HLA determinants expressed by recipient APC. The restriction element of two T cell clones with the HLA phenotype of the first donor (HLA-DR1,8) and one T cell clone with the HLA phenotype of the second transplant (HLA-DR3,9) was HLA-DR4 of the recipient, whereas other T cell clones derived from the second transplant recognized TT in the context of HLA-DR5 of the recipient's APC. These latter T cell clones were not able to proliferate in response to TT when autologous APC were used. These data demonstrate that recipient and donor cells having different HLA phenotypes could cooperate across the allogeneic barrier and that MHC restriction of antigen (Ag) recognition is independent from the MHC genotype of the T cells but is influenced by the environment in which the T cells mature. We also isolated T cell clones that were able to recognize processed TT presented by all allogeneic EBV cell lines tested, indicating that the Ag specificity of these clones was not restricted by a particular class II MHC molecule. The Ag-specific proliferative response of one of these clones could be blocked by anti-class II MHC mAbs. These results demonstrate that in addition to Ag recognition in the context of specific class II MHC Ags, other types of Ag-specific responses may occur in this human chimera. It is not clear whether this "allo" plus Ag recognition is the result of education of transplanted fetal cells in the host thymus. Taking into consideration our previous findings indicating that alloreactive T cell clones specific for the recipient cells could be isolated in vitro from the PBL of the same patient, our data suggest that the mechanism for deletion of self-reactive clones and the generation of MHC-restricted responses are different.     

A lymphokine that activates the cytolytic program of both cytotoxic T lymphocyte and natural killer clones

A 10-12 kD lymphokine, herein termed TCAF, was recently shown to be secreted from Th after crosslinking of their antigen/MHC (T3-Ti) receptors. TCAF stimulates resting T lymphocyte proliferation via binding to surface components of the T11 pathway. To determine whether TCAF could induce antigen-independent activation of the lytic machinery of cytotoxic cells, the present studies were conducted. In the presence of TCAF, both T8+ class I MHC-specific and T4+ class II MHC-specific cytotoxic T cell clones were induced to kill targets, including those lacking the appropriate MHC molecules. This effect was unique to TCAF, since IL-1, IL-2, IFN-gamma could not stimulate lytic activity. Furthermore, both T3+T11+ and T3-T11+ NK clones were triggered to lyse NK-resistant target cells. These findings suggest that TCAF can function in an antigen-independent fashion to amplify cytotoxic effector responses.     

HIV-1 infection leads to increased HLA-E expression resulting in impaired function of natural killer cells

HIV has evolved several strategies to evade recognition by the host immune system including down-regulation of major histocompatibility complex (MHC) class I molecules. However, reduced expression of MHC class I molecules may stimulate natural killer (NK) cell lysis in cells of haematopoietic lineage. Here, we describe how HIV counteracts stimulation of NK cells by stabilizing surface expression of the non-classical MHC class I molecule, HLA-E. We demonstrate enhanced expression of HLA-E on lymphocytes from HIV-infected patients and show that in vitro infection of lymphocytes with HIV results in up-regulation of HLA-E expression and reduced susceptibility to NK cell cytotoxicity. Using HLA-E transfected K-562 cells, we identified the well-known HIV T-cell epitope p24 aa14-22a as a ligand for HLA-E that stabilizes surface expression of HLA-E, favouring inhibition of NK cell cytotoxicity. These results propose HIV-mediated up-regulation of HLA-E expression as an additional evasion strategy targeting the antiviral activities of NK cells, which may contribute to the capability of the virus in establishing chronic infection.     

Mouse CD94/NKG2A Is a Natural Killer Cell Receptor for the Nonclassical Major Histocompatibility Complex (MHC) Class I Molecule Qa-1b

Natural killer (NK) cells preferentially lyse targets that express reduced levels of major histocompatibility complex (MHC) class I proteins. To date, the only known mouse NK receptors for MHC class I belong to the Ly49 family of C-type lectin homodimers. Here, we report the cloning of mouse NKG2A, and demonstrate it forms an additional and distinct class I receptor, a CD94/NKG2A heterodimer. Using soluble tetramers of the nonclassical class I molecule Qa-1^b, we provide direct evidence that CD94/NKG2A recognizes Qa-1^b. We further demonstrate that NK recognition of Qa-1^b results in the inhibition of target cell lysis. Inhibition appears to depend on the presence of Qdm, a Qa-1^b-binding peptide derived from the signal sequences of some classical class I molecules. Mouse NKG2A maps adjacent to CD94 in the heart of the NK complex on mouse chromosome six, one of a small cluster of NKG2-like genes. Our findings suggest that mouse NK cells, like their human counterparts, use multiple mechanisms to survey class I expression on target cells.     

Dendritic cell-lymphoid cell aggregation and major histocompatibility antigen expression during rat cardiac allograft rejection

To determine the pattern of cellular expression of donor MHC class I and class II antigens during the course of rat cardiac allograft rejection, ACI cardiac allografts transplanted to BN recipients were examined from day 2 to day 6 using immunohistologic and immunoelectron microscopic methods. We used both monomorphic and donor-specific mouse anti-rat MHC class I and class II mAbs in this study. In normal ACI hearts, MHC class I reactivity was confined to the vascular endothelium and to interstitial cells. Ongoing rejection was characterized by an increased donor MHC class I staining intensity of microvascular endothelium and induction of donor class I surface reactivity on cardiac myofibers. Donor MHC class II reactivity was exclusively confined to interstitial dendritic cells (IDC) in both normal ACI hearts and in rejecting allografts, although rejection was associated with marked fluctuations in class II IDC frequency. An early numerical depression in class II IDC present in both allografts and syngeneic heart grafts was attributed to a direct effect of the transplantation procedure. By days 3-4, allografts showed an absolute overall increase in donor class II IDC frequency, which was associated with the presence of multiple localized high-density IDC-lymphocyte aggregates. The lymphocytes present in the focal areas were predominantly of the class II-reactive Th cell subpopulation. These aggregates may thus represent the in vivo homologue of dendritic cell-lymphocyte clustering, which has been shown to be required for primary class II allosensitization in the rat and mouse in vitro. During the late phase of rejection, there was a marked numerical fall in donor class II IDC, which correlated with extensive overall graft destruction. This study has shown that acute rat cardiac allograft rejection can occur in the absence of donor MHC class II expression by allograft vascular endothelium and cardiac myofibers. The IDC, which are believed to represent the principal class II alloantigen presenting cells in the rat heart, remain the sole class II-expressing cellular constituents of the graft throughout the course of rejection.     

Re-educating natural killer cells

The development and function of natural killer (NK) cells is dictated by signals received through activating and inhibitory receptors expressed on the cell surface. During their maturation in the bone marrow, NK cells undergo an education process that ensures they are tolerant to healthy peripheral tissues. Several recent studies advance our understanding of self-tolerance mechanisms at work in NK cells. These studies demonstrate that the developmental programming in NK cells is not fixed, and that perturbations to the peripheral environment (via transplantation or viral infection, for example) greatly influence the ability of mature NK cells to mount an effector response. This newfound ability of mature NK cells to be “re-educated” may be clinically applicable in the immunotherapeutic use of NK cells against infection and cancer.The immune system employs intricate mechanisms to maintain self-tolerance; these regulatory mechanisms ensure that immune cells distinguish foreign invaders from healthy tissues. To ensure self-tolerance in T cells, an entire organ (the thymus) is devoted to selecting specific thymocytes that are able to recognize major histocompatability complex (MHC)–self-peptide antigens while deleting thymocytes that recognize these complexes too strongly. Analogous regulatory mechanisms are in place to assure self-tolerance during the development of B cells in the bone marrow. Natural killer (NK) cells are no exception and also undergo an education process during development whereby cells that are potentially self-reactive are rendered anergic.NK cells, which were first described by several groups in the early 1970s (Greenberg et al., 1973; Herberman et al., 1975; Kiessling et al., 1975; Sendo et al., 1975; Zarling et al., 1975), circulate through the blood and lymphatics and reside in virtually all organs. In these tissues, NK cells are poised to eliminate stressed, virally infected, or transformed cells without prior sensitization while minimizing injury to normal healthy cells. In humans and mice, NK cells survey their environment by using a sophisticated repertoire of evolutionarily selected activating and inhibitory receptors that bind both host- and pathogen-encoded ligands (Lanier, 2005). Because NK cells are powerful effector lymphocytes, their activation must be tightly regulated to ensure that NK cells protect the host from pathogen invasion while avoiding deleterious autoimmune responses. Thus, during their development in the bone marrow, NK cells are trained, or “educated,” to distinguish healthy from abnormal tissues.

Education and self-tolerance of NK cells

In 1986, Kärre et al. (1986) observed that unlike T cells, which respond to foreign protein components bound to MHC molecules, NK cells attack cells that are “missing self,” i.e., lacking MHC molecules. The missing self hypothesis inferred that an NK cell had to possess inhibitory receptors that could bind MHC class I (expressed on virtually all healthy cells), thereby preventing the NK cell from becoming activated during normal healthy conditions (Ljunggren and Kärre, 1990). A couple of years later, Karlhofer et al. (1992) provided the first molecular evidence for the missing self hypothesis by identifying an inhibitory receptor (Ly49A on mouse NK cells) that specifically recognizes MHC class I and suppresses NK cell function. At the same time, amino acid residues in human MHC class I that specifically rendered target cells resistant to NK cell–mediated cytotoxicity were identified (Storkus et al., 1991). Soon thereafter, several groups identified and cloned the genes encoding human inhibitory NK cell receptors that recognize different HLA class I family members (Colonna and Samaridis, 1995; D’Andrea et al., 1995; Gumperz et al., 1995; Wagtmann et al., 1995). The responsible inhibitory receptors were designated the killer cell immunoglobulin-like receptor (KIR) family (Long et al., 1996). Later studies demonstrated that in addition to sensing a loss of MHC class I in target cells, full effector function of NK cells requires triggering of their activating receptors via stress-induced or virus-encoded ligands on target cells (Cerwenka et al., 2001; Diefenbach et al., 2001; Arase et al., 2002; Smith et al., 2002). These studies explain the inability of NK cells to attack healthy cells that display either no MHC class I (e.g., erythrocytes) or low levels of MHC class I (e.g., neurons) on their surface. Although NK cells have been studied for several decades, the activating NK cell receptors and ligands responsible for mediating “missing self” rejection of MHC class I–deficient cells remains elusive.During development, NK cells have been shown to transit through several distinct stages, which are defined by acquisition of function and expression of surface receptors (Yokoyama et al., 2004; Di Santo, 2006). Immature mouse NK cells begin to express inhibitory Ly49 receptors early in development; this initiates an education process whereby inhibitory Ly49 receptor engagement of autologous MHC class I results in the generation of functional effector NK cells in the periphery (Fig. 1 A; Fernandez et al., 2005; Kim et al., 2005). This selection process has been termed “licensing” or “arming” of NK cells. Failure to engage inhibitory receptors during development, due to lack of inhibitory receptor expression on the NK cell or lack of interaction with MHC class I, results in the generation of a subset of anergic or hyporesponsive peripheral NK cells (Fig. 1, B and C; Fernandez et al., 2005; Kim et al., 2005). Similarly, human NK cells that express certain inhibitory KIRs that engage cognate HLA during development gain effector function; in the absence of inhibitory KIR–HLA interactions, human NK cells are rendered hyporesponsive (Anfossi et al., 2006; Yu et al., 2007; Kim et al., 2008). The education of NK cells is also influenced by signals received through activating receptors. In a process analogous to negative selection of developing T cells, ligation of activating receptors on developing NK cells by ubiquitiously expressed cognate viral or self-ligands leads to both a repression of cellular function through that particular receptor and a partial deletion of the subset repertoire (Ogasawara et al., 2005; Oppenheim et al., 2005; Sun and Lanier, 2008b; Tripathy et al., 2008). Altogether, these mechanisms are thought to exist to ensure that mature NK cells do not attack healthy self-tissues.Open in a separate windowFigure 1.Education and re-education of NK cells. The figure depicts the “education” of developing NK cells in different bone marrow environments (left) and the experimental “re-education” of mature NK cells in different peripheral environments (right). (A) Immature NK cells expressing inhibitory receptors that engage MHC class I become responsive mature effector cells. (B) Immature NK cells expressing inhibitory receptors that do not engage MHC class I become anergic cells. (C) Immature NK cells lacking inhibitory receptors that can engage MHC class I also become anergic cells. (D) Mature responsive NK cells that are adoptively transferred into a MHC class I–deficient environment become anergic. (E) Mature anergic NK cells (expressing inhibitory receptors for MHC class I) that are adoptively transferred into a MHC class I–sufficient setting become responsive. (F) During viral infection and inflammation, mature anergic NK cells (lacking inhibitory receptors for MHC class I) become activated and hyperresponsive effector cells.

Re-education of NK cells

Although NK cell precursors are primarily found in the bone marrow, there is evidence that NK cells can also develop in peripheral organs, including the thymus (Vosshenrich et al., 2006), and possibly in human lymph nodes (Freud et al., 2005). There is also evidence that NK cells continue to mature after egress from the bone marrow, as splenic NK cell populations consist of cells displaying varying degrees of maturation (which correlate with their expression of markers such as CD27, Mac-1, and KLRG1; Hayakawa and Smyth, 2006; Huntington et al., 2007a; Chiossone et al., 2009). In studies published in this issue, Joncker et al. and Elliot et al. find that the ability of mature NK cells to produce IFN-γ and kill target cells can be reprogrammed after the cells are exposed to MHC class I environments different than the ones in which they developed. In these studies, splenic NK cells that matured in a MHC class I–sufficient environment and acquired full effector function were adoptively transferred into a new host that was devoid of MHC class I. Surprisingly, these mature NK cells became anergic to receptor stimulation within several days after adoptive transfer into the MHC class I–deficient recipient (Fig. 1 D). In the reciprocal experiment, unresponsive NK cells from MHC class I–deficient mice were adoptively transferred into MHC class I–sufficient hosts (either in high numbers, or into an NK cell–deficient host strain to avoid rejection); these transferred cells gained effector function (Fig. 1 E). Like wild-type NK cells, these previously anergic NK cells now produced high levels of IFN-γ and degranulated robustly when various activating receptors were triggered ex vivo.Previous data suggested that the education of NK cells is restricted to the bone marrow (Yokoyama et al., 2004; Huntington et al., 2007b). However, these new studies indicate that developmental programming in NK cells is not entirely fixed, and that mature NK cells can be “re-educated,” gaining or losing functional capacity as their new environment dictates. It will be interesting to learn where the reprogramming is occurring. It remains to be determined whether adoptively transferred mature NK cells re-enter the bone marrow, or are reprogrammed in peripheral tissues. In the clinical setting, the functional plasticity of NK cells could potentially be harnessed for NK cell immunotherapy against certain tumors or viruses, where a robust NK cell response against cells that have down-modulated HLA expression would be efficacious.

Unleashing NK cells

In the steady state, both responsive and anergic NK cells reside in peripheral organs. Why is it that NK cells lacking an inhibitory receptor for autologous MHC class I (“unlicensed” or “disarmed” NK cells) are allowed to seed the periphery? Why are these cells not subject to apoptosis like developing T cells bearing TCRs that cannot properly engage MHC during positive selection? In fact, recent studies suggest that these anergic or unlicensed cells play an important role during viral infection. During mouse cytomegalovirus (MCMV) infection, NK cells in both wild-type and MHC class I–deficient mice mount robust effector responses, and NK cells that were previously tolerant to MHC class I–deficient cells in mixed bone marrow chimeric mice rapidly reject their MHC class I–deficient neighbors (Sun and Lanier, 2008a). In this setting, inflammation was able to break self-tolerance in vivo. These findings are consistent with in vitro experiments demonstrating that anergic NK cells can secrete large amounts of IFN-γ when bathed in proinflammatory cytokines such as interleukin (IL) 12 and IL-18 (Yokoyama and Kim, 2006). Likewise, proinflammatory cytokines can activate anergic T cells (Schwartz, 2003).A recent study surprisingly showed that these unlicensed, anergic NK cells are actually better effectors than licensed NK cells during MCMV infection (Orr et al., 2010). Depletion of unlicensed, but not licensed, NK cells from MCMV-infected wild-type B6 mice resulted in elevated viral titers. Interestingly, adoptively transferred unlicensed NK cells provided robust protection against MCMV challenge in neonate mice, whereas an equal number of licensed NK cells was unable to promote survival any better than the negative control receiving no NK cells. Together, these findings suggest that although inhibitory receptors are required for self-tolerance, they hinder productive immune responses during infection. Thus, an overall lack of inhibitory receptors against autologous MHC class I permits NK cells to respond more robustly against viral infection (Fig. 1 F). Perhaps infection and inflammation stimulate responsive (licensed) and anergic (unlicensed) NK cells similarly, but once the initial threshold for activation is attained, those NK cells without inhibiting receptors intuitively respond faster and more robustly. Although the potential for autoimmunity and greater collateral damage resides in the unlicensed NK cells, it is possible that evolutionary pressures have allowed for the selection and maintenance of this normally anergic subset specifically to deal with infectious pathogens.In addition, these features may prove useful in the setting of allogeneic hematopoietic stem cell transplantation for the treatment of leukemia patients. In several independent studies, patients receiving inhibitory KIR–HLA mismatched transplants had a lower incidence of leukemia relapse and a higher frequency of survival compared with patients receiving NK cell populations that could be inhibited by host HLA molecules (Hsu et al., 2005; Clausen et al., 2007; Miller et al., 2007; Sobecks et al., 2007; Yu et al., 2009). A greater understanding of how NK cells develop and function may advance the prevention and treatment of certain infectious diseases and cancers.     

Autologous stem cell transplant recipients tolerate haploidentical related‐donor natural killer cell–enriched infusions

BACKGROUND: In the setting of allogeneic stem cell transplantation (SCT), infusing natural killer (NK) cells from a major histocompatibility complex (MHC)‐mismatched donor can mediate an antileukemic effect. The graft‐versus‐tumor effect after autologous stem cell transplantation (ASCT) may result in less disease relapse. STUDY DESIGN AND METHODS: We performed a Phase I clinical trial to assess the safety and feasibility of infusing distantly processed donor NK‐enriched mononuclear cell (NK‐MNC) infusions from a MHC haplotype–mismatched (haploidentical) donor to patients who recently underwent ASCT for a hematologic malignancy. On Day 1, peripheral blood MNCs were obtained by steady‐state leukapheresis and sent from Boston to the Production Assistance for Cellular Therapies (PACT) facility at the University of Minnesota, where immunomagnetic depletion of CD3 cells was performed on Day 2. NK‐MNC products were then returned to Boston on Day 2 for infusion on Day 3. Toxicity, cellular product characteristics, and logistic events were monitored. RESULTS: At a median of 90 days (range, 49‐191 days) after ASCT, 13 patients were treated with escalating doses of NK‐MNCs per kilogram from 10⁵ to 2 × 10⁷. Adverse effects included Grade 2 rigors and muscle aches, but no Grade 3 or 4 events and no graft‐versus‐host disease or marrow suppression. One air courier delay occurred. NK‐MNC products were viable with cytotoxic activity after transport. CONCLUSION: CD3‐depleted, MHC‐mismatched allogeneic NK‐MNC infusions can be safely and feasibly administered to patients after ASCT after distant processing and transport, justifying further development of this approach.