Major histocompatibility complex (MHC) class I KbDb −/− deficient mice possess functional CD8+ T cells and natural killer cells

We obtained mice deficient for major histocompatibility complex (MHC) molecules encoded by the H-2K and H-2D genes. H-2 KbDb −/− mice express no detectable classical MHC class I-region associated (Ia) heavy chains, although β2-microglobulin and the nonclassical class Ib proteins examined are expressed normally. KbDb −/− mice have greatly reduced numbers of mature CD8+ T cells, indicating that selection of the vast majority (>90%) of CD8+ T cells cannot be compensated for by β2-microglobulin-associated molecules other than classical H-2K and D locus products. In accord with the greatly reduced number of CD8+ T cells, spleen cells from KbDb −/− mice do not generate cytotoxic responses in primary mixed-lymphocyte cultures against MHC-disparate (allogeneic) cells. However, in vivo priming of KbDb −/− mice with allogeneic cells resulted in strong CD8+ MHC class Ia-specific allogeneic responses. Thus, a minor population of functionally competent peripheral CD8+ T cells capable of strong cytotoxic activity arises in the complete absence of classical MHC class Ia molecules. KbDb −/− animals also have natural killer cells that retain their cytotoxic potential.

The α1 domain of HLA-G1 and HLA-G2 inhibits cytotoxicity induced by natural killer cells: Is HLA-G the public ligand for natural killer cell inhibitory receptors?

We have investigated the protective role of the membrane-bound HLA-G1 and HLA-G2 isoforms against natural killer (NK) cell cytotoxicity. For this purpose, HLA-G1 and HLA-G2 cDNAs were transfected into the HLA class I-negative human K562 cell line, a known reference target for NK lysis. The HLA-G1 protein, encoded by a full-length mRNA, presents a structure similar to that of classical HLA class I antigens. The HLA-G2 protein, deduced from an alternatively spliced transcript, consists of the α1 domain linked to the α3 domain. In this study we demonstrate that (i) HLA-G2 is present at the cell surface as a truncated class I molecule associated with β2-microglobulin; (ii) NK cytolysis, observed in peripheral blood mononuclear cells and in polyclonal CD3− CD16+ CD56+ NK cells obtained from 20 donors, is inhibited by both HLA-G1 and HLA-G2; this HLA-G-mediated inhibition is reversed by blocking HLA-G with a specific mAb; this led us to the conjecture that HLA-G is the public ligand for NK inhibitory receptors (NKIR) present in all individuals; (iii) the α1 domain common to HLA-G1 and HLA-G2 could mediate this protection from NK lysis; and (iv) when transfected into the K562 cell line, both HLA-G1 and HLA-G2 abolish lysis by the T cell leukemia NK-like YT2C2 clone due to interaction between the HLA-G isoform on the target cell surface and a membrane receptor on YT2C2. Because NKIR1 and NKIR2, known to interact with HLA-G, were undetectable on YT2C2, we conclude that a yet-unknown specific receptor for HLA-G1 and HLA-G2 is present on these cells.

High-affinity binding of bioactive glycosylation-inhibiting factor to antigen-primed T cells and natural killer cells

High-affinity binding was demonstrated between suppressor-T-cell-derived bioactive glycosylation-inhibiting factor (GIF) and helper T hybridomas and natural killer cell line cells. Inactive GIF present in cytosol of suppressor T cells and Escherichia coli-derived recombinant human GIF (rhGIF) failed to bind to these cells. However, affinity of rhGIF for the target cells was generated by replacement of Cys-57 in the sequence with Ala or of Asn-106 with Ser or binding of 5-thio-2-nitrobenzoic acid to Cys-60 in the molecule. Such mutations and the chemical modification of rhGIF synergistically increased the affinity of GIF molecules for the target cells. The results indicated that receptors on the target cells recognize conformational structures of bioactive GIF. Equilibrium dissociation constant (Kd) of the specific binding between bioactive rGIF derivatives and high-affinity receptors was 10–100 pM. Receptors for bioactive GIF derivatives were detected on Th1 and Th2 T helper clones and natural killer NK1.1+ cells in normal spleen but not on naive T or B cells. Neither the inactive rGIF nor bioactive rGIF derivatives bound to macrophage and monocyte lines or induced macrophages for tumor necrosis factor α production.

Multiple receptors for HLA-G on human natural killer cells

HLA-G is the putative natural killer (NK) cell inhibitory ligand expressed on the extravillous cytotrophoblast of the human placenta. Killing of the class I negative human B cell line 721.221 by NK cells is inhibited by the expression of HLA-G. This inhibition is dependent on a high level of HLA-G expression. In the present study, the nature of the receptors that mediate the inhibition has been studied with 140 NK cell lines from two donors and 246 NK clones from 5 donors by blocking the inhibition using monoclonal antibodies against the known NK inhibitory receptors: CD158a, CD158b, and CD94. Both CD94 and the two CD158 proteins can function as receptors, although the former clearly predominates. In many cases, a combination of antibodies to these receptors is required to achieve maximal reversal of inhibition. Moreover, in at least one-third of the NK cells that are inhibited by HLA-G, these antibodies alone or in combination do not reverse inhibition, strongly suggesting the existence of a third major unidentified receptor for HLA-G.

Murine Nkg2d and Cd94 are clustered within the natural killer complex and are expressed independently in natural killer cells

Natural killer (NK) cells express C-type lectin-like receptors, encoded in the NK gene complex, that interact with major histocompatibility complex class I and either inhibit or activate functional activity. Human NK cells express heterodimers consisting of CD94 and NKG2 family molecules, whereas murine NK cells express homodimers belonging to the Ly-49 family. The corresponding orthologues for other species, however, have not been described. In this report, we used probes derived from the expressed sequence tag database to clone C57BL/6-derived cDNAs homologous to human NKG2-D and CD94. Among normal tissues, murine NKG2-D and CD94 transcripts are highly expressed only in activated NK cells, including both Ly-49A+ and Ly-49A− subpopulations. Additionally, mNKG2-D is expressed in murine NK cell clones KY-1 and KY-2, whereas mCD94 expression is observed only in KY-1 cells but not KY-2. Last, we have finely mapped the physical location of the Cd94 (centromeric) and Nkg2d (telomeric) genes between Cd69 and the Ly49 cluster in the NK complex. Thus, these data indicate the expanding complexity of the NK complex and the corresponding repertoire of C-type lectin-like receptors on murine NK cells.

Commitment to natural killer cells requires the helix–loop–helix inhibitor Id2

We have previously described how T and natural killer (NK) lineage commitment proceeds from common T/NK progenitors (p-T/NK) in the murine fetal thymus (FT), with the use of a clonal assay system capable of discriminating p-T/NK from unipotent T or NK lineage-committed progenitors (p-T and p-NK, respectively). The molecular mechanisms controlling the commitment processes, however, are yet to be defined. In this study, we investigated the progenitor activity of FT cells from Id2−/− mice that exhibit defective NK cell development. In the Id2−/− FT, NK cells were greatly reduced, and a cell population that exclusively contains p-NK in the wild-type thymus was completely missing. Id2−/− FT progenitors were unable to differentiate into NK cells in IL-2-supplemented-FT organ culture. Single progenitor analysis demonstrated that all Id2−/− fetal thymic progenitors are destined for the T cell lineage, whereas progenitors for T/NK, T, and NK cell lineages were found in the control. Interestingly, the total progenitor number was similar between Id2−/− and Id2+/+ embryos analyzed. Expression of Id2 was correlated with p-NK activity. Our results suggest that Id2 is indispensable in thymic NK cell development, where it most probably restricts bipotent T/NK progenitors to the NK cell lineage.

Characterization of GMP-17, a granule membrane protein that moves to the plasma membrane of natural killer cells following target cell recognition.

Cytotoxic lymphocytes are characterized by their inclusion of cytoplasmic granules that fuse with the plasma membrane following target cell recognition. We previously identified a cytotoxic granule membrane protein designated p15-TIA-1 that is immunochemically related to an RNA-recognition motif (RRM)-type RNA-binding protein designated p40-TIA-1. Although it was suggested that p15-TIA-1 might be derived from p40-T1A-1 by proteolysis, N-terminal amino acid sequencing of p15-TIA-1 immunoaffinity purified from a natural killer (NK) cell line by using monoclonal antibody (mAb) 2G9 revealed that p15-T1A-1 is identical to the deduced amino acid sequence of NKG7 and GIG-1, cDNAs isolated from NK cells and granulocyte-colony-stimulating factor-treated mononuclear cells, respectively. Epitope mapping revealed that mAb 2G9 recognizes the C terminus of p15-T1A-1 and p40-T1A-1. The deduced amino acid sequence of p15-T1A-1/NKG7/GIG-1 predicts that the protein possesses four transmembrane domains, and immuno-electron microscopy localizes the endogenous protein to the membranes of cytotoxic granules in NK cells. Given its subcellular localization, we propose to rename-this protein GMP-17, for granule membrane protein of 17 kDa. Immunofluorescence microscopy of freshly isolated NK cells confirms this granular localization. Target cell-induced NK cell degranulation results in translocation of GMP-17 from granules to the plasma membrane, suggesting a possible role for GMP-17 in regulating the effector function of lymphocytes and neutrophils.

Normal development and function of natural killer cells in CD3 epsilon delta 5/delta 5 mutant mice.

The CD3 epsilon polypeptide contributes to the cell surface display as well as to the signal transduction properties of the T-cell antigen receptor complex. Intriguingly, the distribution of CD3 epsilon is not restricted to T cells, since activated mouse, human, and avian natural killer (NK) cells do express intracytoplasmic CD3 epsilon polypeptides. CD3 epsilon is also present in the cytoplasm of fetal thymic T/NK bipotential progenitor cells, suggesting that it constitutes a component of the NK differentiation program. We report here that the genetic disruption of CD3 epsilon exon 5 alters neither NK cell development nor in vitro and in vivo NK functions, although it profoundly blocked T-cell development. These results support the notion that CD3 epsilon is dispensable for mouse NK cell ontogeny and function and further suggest that the common NK/T-cell progenitor cell utilizes CD3 epsilon as a mandatory component only when differentiating toward the T-cell lineage.

Inhibition of selective signaling events in natural killer cells recognizing major histocompatibility complex class I.

Many studies have characterized the transmembrane signaling events initiated after T-cell antigen receptor recognition of major histocompatibility complex (MHC)-bound peptides. Yet, little is known about signal transduction from a set of MHC class I recognizing receptors on natural killer (NK) cells whose ligation dramatically inhibits NK cell-mediated killing. In this study we evaluated the influence of MHC recognition on the proximal signaling events in NK cells binding tumor targets. We utilized two experimental models where NK cell-mediated cytotoxicity was fully inhibited by the recognition of specific MHC class I molecules. NK cell binding to either class I-deficient or class I-transfected target cells initiated rapid protein tyrosine kinase activation. In contrast, whereas NK cell binding to class I-deficient targets led to inositol phosphate release and increased intracellular free calcium ([Ca2+]i), NK recognition of class I-bearing targets did not induce the activation of these phospholipase C-dependent signaling events. The recognition of class I by NK cells clearly had a negative regulatory effect since blocking this interaction using anti-class I F(ab')2 fragments increased inositol 1,4,5-trisphosphate release and [Ca2+]i and increased the lysis of the targets. These results suggest that one of the mechanisms by which NK cell recognition of specific MHC class I molecules can block the development of cell-mediated cytotoxicity is by inhibiting specific critical signaling events.

Natural killer and lymphokine-activated killer cells require granzyme B for the rapid induction of apoptosis in susceptible target cells.

Granzyme (Gzm) B-deficient mice obtained by gene targeting were used to assess the role of Gzm B in the mechanisms used by natural killer (NK) and lymphokine-activated killer (LAK) cells to destroy target cells. Gzm B-/- NK cells, LAK cells, and cytotoxic T lymphocytes (CTL) all are defective in their ability to rapidly induce DNA fragmentation/apoptosis in susceptible target cells. This defect can be partially corrected with long incubation times of effector and target cells. Moreover, Gzm B-/- NK cells (but not CTL or LAK cells) exhibit a defect in 51Cr release from susceptible target cells. This 51Cr release defect in Gzm B-deficient NK cells is also not overcome by prolonged incubation times or high effector-to-target cell ratios. We conclude that Gzm B plays a critical and nonredundant role in the rapid induction of DNA fragmentation/apoptosis by NK cells, LAK cells, and CTL. Gzm B may have an additional role in NK cells (but not in CTL or LAK cells) for mediating 51Cr release.

Natural killer cell lines kill autologous β2-microglobulin-deficient melanoma cells: Implications for cancer immunotherapy

Cancer vaccines used to generate specific cytotoxic T lymphocytes are not effective against tumor cells that have lost or suppressed expression of their class I major histocompatibility complex proteins. This loss is common in some cancers and particularly in metastatic lesions. We show that β2-microglobulin-deficient class I-negative melanoma variants derived from patients undergoing specific T cell therapy are lysed by heterologous as well as autologous natural killer (NK) lines and clones, but not by specific T cells. Moreover, the minor NK cell fraction but not the major T cell fraction derived from heterologous lymphokine activated killer cells kills those tumor cell lines. ICAM-1 expression by the different class I protein deficient tumors was correlated with their sensitivity to lysis by NK cells. Adoptive autologous NK therapy may be an important supplement to consider in the design of new cancer immunotherapies.

Engagement of p75/AIRM1 or CD33 inhibits the proliferation of normal or leukemic myeloid cells

P75/AIRM1 is a recently identified surface molecule that belongs to the sialoadhesin family and displays homology with the myeloid cell antigen CD33. In lymphoid cells, p75/AIRM1 is confined to natural killer cells and mediates inhibition of their cytolytic activity. In this study, we show that p75/AIRM1 is also expressed by cells of the myelomonocytic cell lineage, in which it appears at a later stage as compared with CD33. In vitro proliferation and differentiation of cord blood-derived CD34+ cells (induced by stem cell factor and granulocyte–macrophage colony-stimulating factor) were consistently inhibited by the addition of anti-p75/AIRM1 mAb. Engagement of CD33 led to inhibition in some experiments. A sharp decrease of cell proliferation/survival was detected in all three p75/AIRM1+ chronic myeloid leukemias analyzed when cultured in the presence of either anti-p75/AIRM1 or anti-CD33 mAbs. Thus, the present study suggests that p75/AIRM1 and CD33 may play a regulatory role in normal myelopoiesis and may be viewed as suitable target molecules to counteract the proliferation/survival of chronic myeloid leukemias.

Priming of tumor-specific T cells in the draining lymph nodes after immunization with interleukin 2-secreting tumor cells: three consecutive stages may be required for successful tumor vaccination.

Although both CD4+ and CD8+ T cells are clearly required to generate long-lasting anti-tumor immunity induced by s.c. vaccination with interleukin 2 (IL-2)-transfected, irradiated M-3 clone murine melanoma cells, some controversy continues about the site and mode of T-cell activation in this system. Macrophages, granulocytes, and natural killer cells infiltrate the vaccination site early after injection into either syngeneic euthymic DBA/2 mice or athymic nude mice and eliminate the inoculum within 48 hr. We could not find T cells at the vaccination site, which argues against the concept that T-cell priming by the IL-2-secreting cancer cells occurs directly at that location. However, reverse transcription-PCR revealed transcripts indicative of T-cell activation and expansion in the draining lymph nodes of mice immunized with the IL-2-secreting vaccine but not in mice vaccinated with untransfected, irradiated M-3 cells. We therefore propose that the antigen-presenting cells, which invade the vaccination site, process tumor-derived antigens and, subsequently, initiate priming of tumor-specific T lymphocytes in lymphoid organs. These findings suggest a three-stage process for the generation of effector T cells after vaccination with IL-2-secreting tumor cells: (i) tumor-antigen uptake and processing at the site of injection by antigen-presenting cells, (ii) migration of antigen-presenting cells into the regional draining lymph nodes, where T-cell priming occurs, and (iii) circulation of activated T cells that either perform or initiate effector mechanisms leading to tumor cell destruction.

Abnormal development of secondary lymphoid tissues in lymphotoxin β-deficient mice

The tumor necrosis factor (TNF) family cytokines lymphotoxin (LT) α and LTβ form heterotrimers that are expressed on the surface of activated lymphocytes and natural killer cells; LTα homotrimers can be secreted as well. Mice with a disrupted LTα gene lack lymph nodes (LN), Peyer’s patches (PP), and follicular dendritic cell (FDC) networks and reveal profound defects of the splenic architecture. However, it is unclear which of these abnormalities is the result of the absence in LTα homotrimers or LTαβ heterotrimers. To distinguish between these two possibilities, a mouse strain deficient in LTβ was created employing Cre/loxP-mediated gene targeting. Mice deficient in LTβ reveal severe defects in organogenesis of the lymphoid system similar to those of LTα−/− mice, except that mesenteric and cervical LN are present in most LTβ-deficient mice. Both LTβ- and LTα-deficient mice show significant lymphocytosis in the circulation and peritoneal cavity and lymphocytic infiltrations in lungs and liver. After immunization, PNA-positive B cell clusters were detected in the splenic white pulp of LTβ-deficient mice, but FDC networks were severely underdeveloped. Collectively, these results indicate that LTα can signal independently from LTβ in the formation of PNA-positive foci in the spleen, and especially in the development of mesenteric and cervical LN.