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Natural killer (NK) cells kill normal and transformed hematopoietic cells that lack expression of major histocompatibility complex (MHC) class I antigens. Lysis of HLA-negative Epstein Barr virus-transformed B lymphoblastoid cell lines (B-LCL) by human NK cell clones can be inhibited by transfection of the target cells with certain HLA-A, -B, or -C alleles. NK cell clones established from an individual demonstrate clonal heterogeneity in HLA recognition and a single NK clone can recognize multiple alleles. We describe a potential human NK cell receptor (NKB1) for certain HLA-B alleles (e.g., HLA-B*5101 and-B*5801) identified by the mAb DX9. NKB1 is a 70-kD glycoprotein that is expressed on a subset of NK cells and NK cell clones. DX9 monoclonal antibody (mAb) specifically inhibits the interaction between NK cell clones and B-LCL targets transfected with certain HLA-B alleles, but does not affect recognition of HLA-A or HLA-C antigens. An individual NK cell clone can independently recognize B-LCL targets transfected with HLA-B or HLA-C antigens; however, DX9 mAb only affects interaction with transfectants expressing certain HLA-B alleles. These findings demonstrate the existence of NK cell receptors involved in the recognition of HLA-B and imply the presence of multiple receptors for MHC on an individual NK clone.

Prior studies using polyclonal populations of natural killer (NK) cells have revealed that expression of certain major histocompatibility complex (MHC) class I molecules on the membrane of normal and transformed hematopoietic target cells can prevent NK cell-mediated cytotoxicity. However, the extent of clonal heterogeneity within the NK cell population and the effect of self versus non-self MHC alleles has not been clearly established. In the present study, we have generated more than 200 independently derived human NK cell clones from four individuals of known human histocompatibility leukocyte antigens (HLA) type. NK clones were analyzed for cytolytic activity against MHC class I-deficient Epstein Barr virus (EBV) transformed B lymphoblastoid cell lines (B-LCL) stably transfected with several HLA-A, -B, or -C genes representing either self or non-self alleles. All NK clones killed the prototypic HLA-negative erythroleukemia K562 and most lysed the MHC class I-deficient C1R and 721.221 B-LCL. Analysis of the panel of HLA-A, -B, and -C transfectants supported the following general conclusions. (a) Whereas recent studies have suggested that HLA-C antigens may be preferentially recognized by NK cells, our findings indicate that 70% or more of all NK clones are able to recognize certain HLA-B alleles and many also recognize HLA-A alleles. Moreover, a single NK clone has the potential to recognize multiple alleles of HLA-A, HLA-B, and HLA-C antigens. Thus, HLA-C is not unique in conferring protection against NK lysis. (b) No simple patterns of HLA specificity emerged. Examination of a large number of NK clones from a single donor revealed overlapping, yet distinct, patterns of reactivity when a sufficiently broad panel of HLA transfectants was examined. (c) Both autologous and allogeneic HLA antigens were recognized by NK clones. There was neither evidence for deletion of NK clones reactive with self alleles nor any indication for an increased frequency of NK clones recognizing self alleles. (d) With only a few exceptions, protection conferred by transfection of HLA alleles into B-LCL was usually not absolute. Rather a continuum from essentially no protection for certain alleles (HLA-A*0201) to very striking protection for other alleles (HLA-B*5801), with a wide range of intermediate effects, was observed. (e) Whereas most NK clones retained a relatively stable HLA specificity, some NK clones demonstrated variable and heterogeneous activity over time. (f) NK cell recognition and specificity cannot be explained entirely by the presence or absence of HLA class I antigens on the target cell.(ABSTRACT TRUNCATED AT 400 WORDS)

Humans are infected with two distinct strains (Type 1 (T1) and Type 2 (T2)) of Epstein-Barr virus (EBV) that differ substantially in their EBNA2 and EBNA 3A/B/C latency genes and the ability to transform B cells in vitro. While most T1 EBV strains contain the "prototype" form of the BZLF1 immediate-early promoter ("Zp-P"), all T2 strains contain the "Zp-V3" variant, which contains an NFAT binding motif and is activated much more strongly by B-cell receptor signalling. Whether B cells infected with T2 EBV are more lytic than cells infected with T1 EBV is unknown. Here we show that B cells infected with T2 EBV strains (AG876 and BL5) have much more lytic protein expression compared to B cells infected with T1 EBV strains (M81, Akata, and Mutu) in both a cord blood-humanized (CBH) mouse model and EBV-transformed lymphoblastoid cell lines (LCLs). Although T2 LCLs grow more slowly than T1 LCLs, both EBV types induce B-cell lymphomas in CBH mice. T1 EBV strains (M81 and Akata) containing Zp-V3 are less lytic than T2 EBV strains, suggesting that Zp-V3 is not sufficient to confer a lytic phenotype. Instead, we find that T2 LCLs express much higher levels of activated NFATc1 and NFATc2, and that cyclosporine (an NFAT inhibitor) and knockdown of NFATc2 attenuate constitutive lytic infection in T2 LCLs. Both NFATc1 and NFATc2 induce lytic EBV gene expression when combined with activated CAMKIV (which is activated by calcium signaling and activates MEF2D) in Burkitt Akata cells. Together, these results suggest that B cells infected with T2 EBV are more lytic due to increased activity of the cellular NFATc1/c2 transcription factors in addition to the universal presence of the Zp-V3 form of BZLF1 promoter., Competing Interests: The authors have declared that no competing interests exist.

EBV causes human B-cell lymphomas and transforms B cells in vitro. EBNA3C, an EBV protein expressed in latently-infected cells, is required for EBV transformation of B cells in vitro. While EBNA3C undoubtedly plays a key role in allowing EBV to successfully infect B cells, many EBV+ lymphomas do not express this protein, suggesting that cellular mutations and/or signaling pathways may obviate the need for EBNA3C in vivo under certain conditions. EBNA3C collaborates with EBNA3A to repress expression of the CDKN2A-encoded tumor suppressors, p16 and p14, and EBNA3C-deleted EBV transforms B cells containing a p16 germline mutation in vitro. Here we have examined the phenotype of an EBNAC-deleted virus (Δ3C EBV) in a cord blood-humanized mouse model (CBH). We found that the Δ3C virus induced fewer lymphomas (occurring with a delayed onset) in comparison to the wild-type (WT) control virus, although a subset (10/26) of Δ3C-infected CBH mice eventually developed invasive diffuse large B cell lymphomas with type III latency. Both WT and Δ3C viruses induced B-cell lymphomas with restricted B-cell populations and heterogeneous T-cell infiltration. In comparison to WT-infected tumors, Δ3C-infected tumors had greatly increased p16 levels, and RNA-seq analysis revealed a decrease in E2F target gene expression. However, we found that Δ3C-infected tumors expressed c-Myc and cyclin E at similar levels compared to WT-infected tumors, allowing cells to at least partially bypass p16-mediated cell cycle inhibition. The anti-apoptotic proteins, BCL2 and IRF4, were expressed in Δ3C-infected tumors, likely helping cells avoid c-Myc-induced apoptosis. Unexpectedly, Δ3C-infected tumors had increased T-cell infiltration, increased expression of T-cell chemokines (CCL5, CCL20 and CCL22) and enhanced type I interferon response in comparison to WT tumors. Together, these results reveal that EBNA3C contributes to, but is not essential for, EBV-induced lymphomagenesis in CBH mice, and suggest potentially important immunologic roles of EBNA3C in vivo., Competing Interests: The authors have declared that no competing interests exist.

Invariant Natural Killer T (iNKT) cells use highly restricted αβ T cell receptors (TCRs) to probe the repertoire of lipids presented by CD1d molecules. Here, we describe our studies of lysophosphatidylcholine (LPC) presentation by human CD1d and its recognition by a native, LPC-specific iNKT TCR. Human CD1d presenting LPC adopts an altered conformation from that of CD1d presenting glycolipid antigens, with a shifted α1 helix resulting in an open A' pocket. Binding of the iNKT TCR requires a 7-Å displacement of the LPC headgroup but stabilizes the CD1d-LPC complex in a closed conformation. The iNKT TCR CDR loop footprint on CD1d-LPC is anchored by the conserved positioning of the CDR3α loop, whereas the remaining CDR loops are shifted, due in part to amino-acid differences in the CDR3β and Jβ segment used by this iNKT TCR. These findings provide insight into how lysophospholipids are presented by human CD1d molecules and how this complex is recognized by some, but not all, human iNKT cells.

CD1d-mediated presentation of glycolipid antigens to T cells is capable of initiating powerful immune responses that can have a beneficial impact on many diseases. Molecular analyses have recently detailed the lipid antigen recognition strategies utilized by the invariant Vα24-Jα18 TCR rearrangements of iNKT cells, which comprise a subset of the human CD1d-restricted T cell population. In contrast, little is known about how lipid antigens are recognized by functionally distinct CD1d-restricted T cells bearing different TCRα chain rearrangements. Here we present crystallographic and biophysical analyses of α-galactosylceramide (α-GalCer) recognition by a human CD1d-restricted TCR that utilizes a Vα3.1-Jα18 rearrangement and displays a more restricted specificity for α-linked glycolipids than that of iNKT TCRs. Despite having sequence divergence in the CDR1α and CDR2α loops, this TCR employs a convergent recognition strategy to engage CD1d/αGalCer, with a binding affinity (∼2 µM) almost identical to that of an iNKT TCR used in this study. The CDR3α loop, similar in sequence to iNKT-TCRs, engages CD1d/αGalCer in a similar position as that seen with iNKT-TCRs, however fewer actual contacts are made. Instead, the CDR1α loop contributes important contacts to CD1d/αGalCer, with an emphasis on the 4'OH of the galactose headgroup. This is consistent with the inability of Vα24- T cells to respond to α-glucosylceramide, which differs from αGalCer in the position of the 4'OH. These data illustrate how fine specificity for a lipid containing α-linked galactose is achieved by a TCR structurally distinct from that of iNKT cells., Competing Interests: The authors have declared that no competing interests exist.

CD1 molecules are glycoproteins that present lipid antigens at the cell surface for immunological recognition by specialized populations of T lymphocytes. Prior experimental data suggest a wide variety of lipid species can bind to CD1 molecules, but little is known about the characteristics of cellular ligands that are selected for presentation. Here we have molecularly characterized lipids bound to the human CD1d isoform. Ligands were eluted from secreted CD1d molecules and separated by normal phase HPLC, then characterized by mass spectroscopy. A total of 177 lipid species were molecularly identified, comprising glycerophospholipids and sphingolipids. The glycerophospholipids included common diacylglycerol species, reduced forms known as plasmalogens, lyso-phospholipids (monoacyl species), and cardiolipins (tetraacyl species). The sphingolipids included sphingomyelins and glycosylated forms, such as the ganglioside GM3. These results demonstrate that human CD1d molecules bind a surprising diversity of lipid structures within the secretory pathway, including compounds that have been reported to play roles in cancer, autoimmune diseases, lipid signaling, and cell death.

CD1d molecules bind lipid antigens in the endocytic pathway, and access to the pathway is important for the development of CD1d-restricted natural killer T (NKT) cells. Saposins, derived from a common precursor, prosaposin, are small, heat-stable lysosomal glycoproteins required for lysosomal degradation of sphingolipids. Expression of prosaposin is required for efficient lipid binding and recognition of human CD1d molecules by NKT cells. Despite high sequence homology among the four saposins, they have different specificities for lipid substrates and different mechanisms of action. To determine the saposins involved in promoting lipid binding to CD1d, we expressed prosaposin deletion mutants lacking individual saposins in prosaposin-negative, CD1d-positive cells. No individual saposin proved to be absolutely essential, but the absence of saposin B resulted in the lowest recognition of alpha-galactosylceramide by NKT cells. When recombinant exogenous saposins were added to the prosaposin-negative cells, saposin B was the most efficient in restoring CD1d recognition. Saposin B was also the most efficient in mediating alpha-galactosylceramide binding to recombinant plate-bound CD1d and facilitating NKT cell activation. Saposin B could also mediate lipid binding to soluble CD1d molecules in a T cell-independent assay. The optimal pH for saposin B-mediated lipid binding to CD1d, pH 6, is higher than that of lysosomes, suggesting that saposin B may facilitate lipid binding to CD1d molecules throughout the endocytic pathway.

Although invariant NKT (iNKT) cells participate in many aspects of immune responses, the molecular mechanisms regulating their development, maturation, and activation are still poorly understood. GATA-3 is a T cell-specific transcription factor that is also expressed in iNKT cells. The critical role of GATA-3 in conventional alphabeta T cells has been well documented, but whether GATA-3 also regulates the development and function of iNKT cells is unknown. In the present study, we report that deficiency of GATA-3 results in cell-intrinsic defects in the thymic development and peripheral maturation of murine iNKT cells. In addition, GATA-3 is also required for survival, activation, and effector functions of this unique population of T cells. Our data also reveal a previously unidentified peripheral maturation step that is GATA-3 dependent.

In addition to NK T cells expressing invariant Valpha14 or Valpha24 T cell receptors (TCRs), the CD1d-restricted T cell repertoire is comprised of T cells with diverse TCRs that mediate inflammation during autoimmune and infectious disease. Here we describe the isolation of human Valpha24(-) T cells that are activated by antigen and CD1d. Mass spectrometric and NMR studies revealed that the stimulatory compounds were neither peptidic nor lipidic but instead were composed of sulfur and aromatic hydrocarbon rings, corresponding to the general structure of phenyl pentamethyldihydrobenzofuran sulfonates. Studies of the molecular mechanism of T cell activation showed that a clonotypic Valpha2/Vbeta21 TCR transmitted activating signals, which were highly specific for hydroxylation and methylation patterns at the terminal structures of stimulatory compounds. These studies provide evidence for noninvariant CD1d-restricted T cells in humans and identify the complete molecular structure of a nonlipidic small molecule that activates T cells through an alphabeta TCR.

Little is known about the antigen specificity of CD1d-restricted T cells, except that they frequently recognize CD1d-expressing antigen-presenting cells in the absence of exogenous antigen. We previously demonstrated that the 24.8.A iNKT cell hybridoma was broadly reactive with CD1d-transfected cell lines and recognized the polar lipid fraction of a tumor cell extract. In the present study, the antigen recognized by the 24.8.A iNKT cell hybridoma was purified to homogeneity and identified as palmitoyl-oleoyl-sn-glycero-3-phosphoethanolamine (16:0-18:1 PE). The 24.8.A iNKT cell hybridoma recognized synthetic 16:0-18:1[cis] PE, confirming that this phospholipid is antigenic. Recognition correlated with the degree of unsaturation of the acyl chains. Using a panel of synthetic PEs, the 24.8.A iNKT cell hybridoma was shown to be activated by PEs that contained at least one unsaturated acyl chain. The configuration of the double bonds was important, as the 24.8.A iNKT cell hybridoma recognized unsaturated acyl chains in the cis, but not the trans, configuration. PEs with multiple double bonds were recognized better than those with a single double bond, and increasing acyl chain unsaturation correlated with increased binding of PE to CD1d. These data illustrate the potential importance of the acyl chain structure for phospholipid antigen binding to CD1d.

Following inoculation of Ag into the anterior chamber (a.c.), systemic tolerance develops that is mediated in part by Ag-specific efferent CD8(+) T regulatory (Tr) cells. This model of tolerance is called a.c.-associated immune deviation. The generation of the efferent CD8(+) Tr cell in a.c.-associated immune deviation is dependent on IL-10-producing, CD1d-restricted, invariant Valpha14(+) NKT (iNKT) cells. The iNKT cell subpopulations are either CD4(+) or CD4(-)CD8(-) double negative. This report identifies the subpopulation of iNKT cells that is important for induction of the efferent Tr cell. Because MHC class II(-/-) (class II(-/-)) mice generate efferent Tr cells following a.c. inoculation, we conclude that conventional CD4(+) T cells are not needed for the development of efferent CD8(+) T cells. Furthermore, Ab depletion of CD4(+) cells in both wild-type mice (remove both conventional and CD4(+) NKT cells) and class II(-/-) mice (remove CD4(+) NKT cells) abrogated the generation of Tr cells. We conclude that CD4(+) NKT cells, but not the class II molecule or conventional CD4(+) T cells, are required for generation of efferent CD8(+) Tr cells following Ag introduction into the eye. Understanding the mechanisms that lead to the generation of efferent CD8(+) Tr cells may lead to novel immunotherapy for immune inflammatory diseases.

Recent results strengthen evidence that CD1-restricted T cells play important roles in host defense against microbial infections. Human subjects recently infected with Mycobacterium tuberculosis showed elevated responses to CD1c-mediated presentation of a microbial lipid antigen, compared with control donors. Activation of CD1d-restricted NKT cells with a synthetic glycolipid antigen results in improved immune responses to several infectious pathogens.

NKT cells are associated with immunological control of autoimmune disease and cancer and can recognize cell surface mCD1d without addition of exogenous antigens. Cellular antigens presented by mCD1d have not been identified, although NKT cells can recognize a synthetic glycolipid, alpha-GalCer. Here we show that after addition of a lipid extract from a tumor cell line, plate-bound mCD1d molecules stimulated an NKT cell hybridoma. This hybridoma also responded strongly to three purified phospholipids, but failed to recognize alpha-GalCer. Seven of sixteen other mCD1d restricted hybridomas also showed a response to certain purified phospholipids. These findings suggest NKT cells can recognize cellular antigens distinct from alpha-GalCer and identify phospholipids as potential self-antigens presented by mCD1d.

Cytotoxicity of human NK cells is under negative control of killer cell Ig-like receptors (KIR) specific for HLA class I. To determine the specificity of five KIR containing two Ig domains (KIR2D), direct binding of soluble recombinant KIR2D to a panel of HLA class I transfectants was assayed. One soluble KIR2D, derived from an inhibitory receptor with a long cytoplasmic tail (KIR2DL1), bound to HLA-C allotypes containing asparagine 77 and lysine 80 in the heavy chain, as expected, since these allotypes inhibit lysis by NK cells expressing KIR2DL1. Surprisingly, another KIR2D (KIR2DL2), which inhibits NK lysis of cells expressing HLA-C molecules with serine 77 and asparagine 80, bound to HLA-C allotypes carrying either amino acid motif. Expression of the KIR2DL receptors in NK cells using recombinant vaccinia viruses confirmed these patterns of recognition, and identified KIR2DL3 as another KIR reacting with both groups of HLA-C allotypes. Mutagenesis of amino acid 44 in KIR2DL1 and KIR2DL2 suggested this residue controls the affinity of KIR for the 77/80 motif of HLA-C molecules. Two other soluble KIR2D, derived from noninhibitory receptors with short cytoplasmic tails (KIR2DS), did not bind to any of the HLA class I allotypes tested. One of these receptors (KIR2DS2) is closely related in sequence to KIR2DL2. Substitution of tyrosine 45 with the phenylalanine conserved in other KIR was sufficient to permit specific binding of KIR2DS2 to HLA-C. These results show that KIR2DL receptors are specific for HLA-C, but that recognition of HLA-C allotypes appears more permissive than indicated by previous functional experiments.

Whereas the human killer cell inhibitory receptors (KIRs) for HLA class I are immunoglobulin-like monomeric type I glycoproteins, the murine Ly49 receptors for H-2 are type II homodimers of the C-type lectin superfamily. Here, we demonstrate that human NK cells also express C-type lectin receptors that influence recognition of polymorphic HLA-A, HLA-B, and HLA-C molecules. These receptors are heterodimers composed of CD94 chains covalently associated with novel tyrosine-phosphorylated glycoproteins (94AP). Some NK clones recognize a common HLA-C ligand using both KIRs and CD94-94AP receptors. These findings suggest the existence of human inhibitory MHC class I receptors of the immunoglobulin and C-type lectin superfamilies and indicate overlap in ligand specificity.

Natural killer (NK) cells are controlled by receptors specific for polymorphic determinants of class I molecules of the major histocompatibility complex (MHC). The contrasting properties of NK and cytotoxic T cell (CTL) class I receptors provide complementarity in the cytolytic lymphocyte response to viruses, tumours and transplants. Whereas human NK cell class I receptors consist of immunoglobulin domains, their mouse counterparts resemble C-type lectins. This difference may reflect the receptors' diverse and rapidly evolving class I ligands.

Bacterial superantigens bind with high affinity to major histocompatibility complex (MHC) class II antigens on antigen-presenting cells and with T cell antigen receptor (TCR) beta chains on T lymphocytes, which results in the T cell activation responsible for toxic shock syndrome and food poisoning. Many cytotoxic T lymphocyte (CTL) clones were shown to have receptors for human leukocyte antigen (HLA) class I molecules that inhibited superantigen-induced cytotoxicity against appropriate class I-bearing target cells. One type of inhibitory receptor, NKB1, was present on CD4+ and CD8+TCR alpha beta+ CTL clones and blocked the killing of staphylococcal enterotoxin B (SEB)-coated targets bearing certain polymorphic HLA-B molecules. Expression of HLA-A, -B, and -C molecules on the SEB-coated targets also protected against cytolysis mediated by many NKB1-negative T cell clones, suggesting the presence of additional inhibitory MHC class I receptors. These HLA class I receptors may limit tissue destruction and possibly autoimmunity caused by activated T lymphocytes.

HLA haplotypes containing the HLA-B46 allele react with both anti-Cw1 and anti-Cw3 alloantisera, a pattern of reactivity defined as the Cw11 antigen and postulated to involve either a distinctive Cw11 allele or a duplicated HLA-C locus. From serological characterization of CIR cells transfected with B46 cDNA we now demonstrate that the anti-Cw3 reactivity with these haplotypes is solely due to the B46 molecule and not to an HLA-C molecule. Furthermore, isolation and characterization of HLA-C mRNA from cells expressing B46 strongly suggest that anti-Cw1 reactions are directed against the product of a conventional Cw1 allele. The antigenic cross-reactivities of B46 with B62 and Cw3 correlate with its chimaeric primary structure, which is identical to that of B62, except in the alpha 1 helix where it is identical to both Cw3 and Cw1. The structure, distribution and genetic linkage of B46 indicate it is of recent, Asian origin and is the result of a gene conversion, involving Cw1 as the donor gene and B62 as the recipient. These results demonstrate that the Cw11 antigen neither corresponds to a novel HLA-C allele nor a duplicated HLA-C locus, but to a combination of epitopes contributed by linked Cw1 and B46 alleles. The nucleotide sequence we previously and erroneously attributed to a distinct Cw11 allele is now demonstrated to encode Cw8. Isolation of the cDNA clone with this sequence from a library made from a cell homozygous for the B46 haplotype was probably an artefact of contamination.

A sensitive sandwich immunoassay for human chorionic gonadotropin (hCG) was developed with biotin-mediated filtration capture and silicon sensor detection. A high density of biotin on the membrane assured efficient capture of complexes containing streptavidin and analyte. Capture efficiency was not affected over a wide range of filtration flow rates or biotin concentrations. The assay utilized the pH sensing ability of the light addressable potentiometric sensor (LAPS) for the detection of urease-antibody conjugates. A LAPS reader was constructed which allowed the enzyme conjugate to be detected in approximately 1 microliter volumes. Effects from variations in detection volume were studied. 10 pg of hCG could be detected in an assay time of 20 min with four standard deviations separation from background. Comparison to a commercial RIA was made.