Quantification of low affinity binding interactions between natural killer cell inhibitory receptors and targeting ligands with a self-induced back-action actuated nanopore electrophoresis (SANE) sensor.

A plasmonic nanopore sensor enabling detection of bimodal optical and electrical molecular signatures was fabricated and tested for its ability to characterize low affinity ligand-receptor interactions. This plasmonic nanosensor uses self-induced back-action (SIBA) for optical trapping to enable SIBA-actuated nanopore electrophoresis (SANE) through a nanopore located immediately below the optical trap volume. A natural killer (NK) cell inhibitory receptor heterodimer molecule CD94/NKG2A was synthesized to target a specific peptide-presenting Qa-1b Qdm ligand as a simplified model of low-affinity interactions between immune cells and peptide-presenting cancer cells that occurs during cancer immunotherapy. A cancer-irrelevant Qa-1b GroEL ligand was also targeted by the same receptor as a control experiment to test for non-specific binding. The analysis of different pairs of bimodal SANE sensor signatures enabled discrimination of ligand, receptor and their complexes and enabled differentiating between specific and non-specific ligand interactions. We were able to detect ligand-receptor complex binding at concentrations over 500 times lower than the free solution equilibrium binding constant (K D ). Additionally, SANE sensor measurements enabled estimation of the fast dissociation rate (k off) for this low-affinity specific ligand-receptor system, previously shown to be challenging to quantify with commercial technologies. The k off value of targeted peptide-presenting ligands is known to correlate with the subsequent activation of immune cells in vivo, suggesting the potential utility of the SANE senor as a screening tool in cancer immunotherapy.

MHC Molecules, T cell Receptors, Natural Killer Cell Receptors, and Viral Immunoevasins-Key Elements of Adaptive and Innate Immunity.

Molecules encoded by the Major Histocompatibility Complex (MHC) bind self or foreign peptides and display these at the cell surface for recognition by receptors on T lymphocytes (designated T cell receptors-TCR) or on natural killer (NK) cells. These ligand/receptor interactions govern T cell and NK cell development as well as activation of T memory and effector cells. Such cells participate in immunological processes that regulate immunity to various pathogens, resistance and susceptibility to cancer, and autoimmunity. The past few decades have witnessed the accumulation of a huge knowledge base of the molecular structures of MHC molecules bound to numerous peptides, of TCRs with specificity for many different peptide/MHC (pMHC) complexes, of NK cell receptors (NKR), of MHC-like viral immunoevasins, and of pMHC/TCR and pMHC/NKR complexes. This chapter reviews the structural principles that govern peptide/MHC (pMHC), pMHC/TCR, and pMHC/NKR interactions, for both MHC class I (MHC-I) and MHC class II (MHC-II) molecules. In addition, we discuss the structures of several representative MHC-like molecules. These include host molecules that have distinct biological functions, as well as virus-encoded molecules that contribute to the evasion of the immune response.

Function and expression of CD1d and invariant natural killer T-cell receptor in the cotton rat (Sigmodon hispidus).

The cotton rat (Sigmodon hispidus) belongs to the rodent family of Cricetidae and provides a powerful model to study the pathogenesis of human respiratory viruses and measles virus. Recent studies in other rodent models have suggested a role for invariant natural killer T (iNKT) cells in antiviral immunity and vaccination against respiratory virus infections. Using new experimental tools, we provide the first evidence for a functional CD1d cell molecule (crCD1d) and iNKT T-cell receptor in cotton rats. The crCD1d cDNA sequence was identified and crCD1d transductants showed that monoclonal antibody WTH-2 stains crCD1d as efficiently as mouse or rat CD1d. The expression of crCD1d was clearly weaker for thymocytes and B cells, and higher for T cells, which is different to what is found in murine species. The antigen-presenting capacity of crCD1d was demonstrated with crCD1d-immunoglobulin dimers loaded with the glycolipid PBS57, which bound iNKT T-cell receptors. Evidence for functional cotton rat iNKT cells was provided by detection of interferon-γ and interleukin-4 in cultures of splenocytes stimulated with PBS57 and α-galactosylceramide and by specific staining of about 0·2% of splenocytes with PBS57-loaded crCD1d dimers. Canonical AV14/AJ18 rearrangements were identified and found to contain multiple members of the AV14 (AV11) family. One of them was expressed and found to bind CD1d dimers. In summary, these data provide the first evidence for functional CD1d molecules and iNKT T-cell receptors in cotton rats and provide the tools to analyse them both in the cotton rat model of infectious diseases.

Structural insights into activation of antiviral NK cell responses.

Natural killer (NK) cells are key components of innate immune responses, providing surveillance against cells undergoing tumorigenesis or infection, by viruses or internal pathogens. NK cells can directly eliminate compromised cells and regulate downstream responses of the innate and acquired immune systems through the release of immune modulators (cytokines, interferons). The importance of the role NK cells play in immune defense was demonstrated originally in herpes viral infections, usually mild or localized, which become severe and life threatening in NK-deficient patients . NK cell effector functions are governed by balancing opposing signals from a diverse array of activating and inhibitory receptors. Many NK receptors occur in paired activating and inhibitory isoforms and recognize major histocompatibility complex (MHC) class I proteins with varying degrees of peptide specificity. Structural studies have made considerable inroads into understanding the molecular mechanisms employed to broadly recognize multiple MHC ligands or specific pathogen-associated antigens and the strategies employed by viruses to thwart these defenses. Although many details of NK development, signaling, and integration remain mysterious, it is clear that NK receptors are key components of a system exquisitely tuned to sense any dysregulation in MHC class I expression, or the expression of certain viral antigens, resulting in the elimination of affected cells.

Molecular evidence for natural killer-like cells in equine endometrial cups.

OBJECTIVES: To identify equine orthologs of major NK cell marker genes and utilize them to determine whether NK cells are present among the dense infiltration of lymphocytes that surround the endometrial cup structures of the horse placenta during early pregnancy. STUDY DESIGN: PCR primers were developed to detect the equine orthologs of NKP46, CD16, CD56, and CD94; gene expression was detected in RNA isolated from lymphocytes using standard 2-step reverse transcriptase (RT) PCR and products were cloned and sequenced. Absolute real-time RT-PCR was used to quantitate gene expression in total, CD3+, and CD3- peripheral lymphocytes, and invasive trophoblast. Lymphocytes surrounding the endometrial cups (ECL) of five mares in early pregnancy were isolated and NK marker gene expression levels were assayed by quantitative RT-PCR. MAIN OUTCOME MEASURES: Absolute mRNA transcript numbers were determined by performing quantitative RT-PCR and comparing values to plasmid standards of known quantities. RESULTS: NKP46 gene expression in peripheral CD3- lymphocytes was higher than in CD3+ lymphocytes, CD16 levels were higher in the CD3+ population, and no significant differences were detected for CD56 and CD94 between the two groups. Expression of all four NK cell markers was significantly higher in lymphocytes isolated from the endometrial cups of pregnant mares compared to PBMC isolated from the same animal on the same day (NKP46, 14-fold higher; CD94, 8-fold higher; CD16, 20-fold higher; CD56, 44-fold higher). CONCLUSIONS: These data provide the first evidence for the expression of major NK cell markers by horse cells and an enrichment of NK-like cells in the equine endometrium during pregnancy.

Natural killer cell receptors.

Natural killer (NK) cells are an important arm of the innate immune response that are directly involved in the recognition and lysis of virus-infected and tumor cells. Such function is under the control of a complex array of germline-encoded receptors able to deliver either inhibitory or activating signals. The majority of inhibitory receptors expressed by NK cells are major histocompatibility complex (MHC) class I-specific and display clonal and stochastic distribution on the cell surface. Thus, a given NK cell expresses at least one self class I inhibitory receptor. Under normal conditions, the strength of inhibitory signals delivered by multiple interactions always overrides the activating signals, resulting in NK cell self-tolerance. Under certain pathological conditions, such as viral infections or tumor transformation, the delicate balance of inhibition versus activation is broken, resulting in downregulation or loss of MHC class I expression. In general, the degree of inhibition induced by class I-specific receptors is proportional to the amount of these molecules on the cell surface. Thus, in transformed cells, this inhibition can be overridden by the triggering signal cascades, leading to cell activation. The majority of triggering receptors expressed by NK cells belong to the multichain immune recognition receptor (MIRR) family and use separate signal-transducing polypeptides similar to those used by other immune receptors such as the T-cell antigen receptor, the B-cell antigen receptor and other receptors expressed by myeloid cells. Inhibitory receptors are not members of the MIRR family but they are relevant for a better understanding the exquisite equilibrium and regulatory crosstalk between positive and negative signals.