Eric O. Long

Recruitment of Tyrosine Phosphatase HCP by the Killer Cell Inhibitory Receptor

Cytolysis of target cells by natural killer (NK) cells and by some cytotoxic T cells occurs unless prevented by inhibitory receptors that recognize MHC class I on target cells. Human NK cells express a p58 inhibitory receptor specific for HLA-C. We report association of the tyrosine phosphatase HCP with the p58 receptor in NK cells. HCP association was dependent on tyrosine phosphorylation of p58. Phosphotyrosyl peptides corresponding to the p58 tail bound and activated HCP in vitro. Furthermore, introduction of an inactive mutant HCP into an NK cell line prevented the p58-mediated inhibition of target cell lysis. These data imply that the inhibitory function of p58 is dependent on its tyrosine phosphorylation and on recruitment and activation of HCP.

Molecular clones of the p58 NK cell receptor reveal immunoglobulin-related molecules with diversity in both the extra- and intracellular domains

Recognition of major histocompatibility class I molecules on target cells by natural killer (NK) cells confers selective protection from NK-mediated lysis. Cross-linking of the p58 NK receptor, involved in the recognition of HLA-C alleles, delivers a negative signal that prevents target cell lysis. Molecular cloning of the p58 NK receptor reported here revealed a new member of the immunoglobulin superfamily. Five distinct p58 receptors, with sequence diversity in the immunoglobulin-related domains, were identified in a single individual. All NK clones tested expressed at least one p58 member. Three different types of transmembrane and cytoplasmic domains exist, even among receptors with closely related extracellular domains. These data revealed a repertoire of NK cells with clonally distributed p58 receptors exhibiting diversity in both extracellular and intracellular domains.

Essential Role of LAT in T Cell Development

The linker molecule LAT is a substrate of the tyrosine kinases activated following TCR engagement. Phosphorylated LAT binds many critical signaling molecules. The central role of this molecule in TCR-mediated signaling has been demonstrated by experiments in a LAT-deficient cell line. To probe the role of LAT in T cell development, the LAT gene was disrupted by targeting. LAT-deficient mice appeared healthy. Flow cytometric analysis revealed normal B cell populations but the absence of any mature peripheral T cells. Intrathymic development was blocked within the CD4- CD8- stage. No gross abnormality of NK or platelet function was observed. LAT is thus critical to both T cell activation and development.

Controlling Natural Killer Cell Responses: Integration of Signals for Activation and Inhibition

Understanding how signals are integrated to control natural killer (NK) cell responsiveness in the absence of antigen-specific receptors has been a challenge, but recent work has revealed some underlying principles that govern NK cell responses. NK cells use an array of innate receptors to sense their environment and respond to alterations caused by infections, cellular stress, and transformation. No single activation receptor dominates; instead, synergistic signals from combinations of receptors are integrated to activate natural cytotoxicity and cytokine production. Inhibitory receptors for major histocompatibility complex class I (MHC-I) have a critical role in controlling NK cell responses and, paradoxically, in maintaining NK cells in a state of responsiveness to subsequent activation events, a process referred to as licensing. MHC-I-specific inhibitory receptors both block activation signals and trigger signals to phosphorylate and inactivate the small adaptor Crk. These different facets of inhibitory signaling are incorporated into a revocable license model for the reversible tuning of NK cell responsiveness.

Activation, coactivation, and costimulation of resting human natural killer cells

Summary:  Natural killer (NK) cells possess potent perforin‐ and interferon‐γ‐dependent effector functions that are tightly regulated. Inhibitory receptors for major histocompatibility complex class I display variegated expression among NK cells, which confers specificity to individual NK cells. Specificity is also provided by engagement of an array of NK cell activation receptors. Target cells may express ligands for a multitude of activation receptors, many of which signal through different pathways. How inhibitory receptors intersect different signaling cascades is not fully understood. This review focuses on advances in understanding how activation receptors cooperate to induce cytotoxicity in resting NK cells. The role of activating receptors in determining specificity and providing redundancy of target cell recognition is discussed. Using Drosophila insect cells as targets, we have examined the contribution of individual receptors. Interestingly, the strength of activation is not determined simply by additive effects of parallel activation pathways. Combinations of signals from different receptors can have different outcomes: synergy, no enhancement over individual signals, or additive effects. Cytotoxicity requires combined signals for granule polarization and degranulation. The integrin leukocyte function‐associated antigen‐1 contributes a signal for polarization but not for degranulation. Conversely, CD16 alone or in synergistic combinations, such as NKG2D and 2B4, signals for phospholipase‐C‐γ‐ and phosphatidylinositol‐3‐kinase‐dependent degranulation.

Regulation of human NK-cell cytokine and chemokine production by target cell recognition

Natural killer (NK)-cell recognition of infected or neoplastic cells can induce cytotoxicity and cytokine secretion. So far, it has been difficult to assess the relative contribution of multiple NK-cell activation receptors to cytokine and chemokine production upon target cell recognition. Using Drosophila cells expressing ligands for the NK-cell receptors LFA-1, NKG2D, DNAM-1, 2B4, and CD16, we studied the minimal requirements for secretion by freshly isolated, human NK cells. Target cell stimulation induced secretion of predominately proinflammatory cytokines and chemokines. Release of chemokines MIP-1alpha, MIP-1beta, and RANTES was induced within 1 hour of stimulation, whereas release of TNF-alpha and IFN-gamma occurred later. Engagement of CD16, 2B4, or NKG2D sufficed for chemokine release, whereas induction of TNF-alpha and IFN-gamma required engagement of additional receptors. Remarkably, our results revealed that, upon target cell recognition, CD56(dim) NK cells were more prominent cytokine and chemokine producers than CD56(bright) NK cells. The present data demonstrate how specific target cell ligands dictate qualitative and temporal aspects of NK-cell cytokine and chemokine responses. Conceptually, the results point to CD56(dim) NK cells as an important source of cytokines and chemokines upon recognition of aberrant cells, producing graded responses depending on the multiplicity of activating receptors engaged.

Cytolytic granule polarization and degranulation controlled by different receptors in resting NK cells

The relative contribution to cytotoxicity of each of the multiple NK cell activation receptors has been difficult to assess. Using Drosophila insect cells, which express ligands of human NK cell receptors, we show that target cell lysis by resting NK cells is controlled by different receptor signals for cytolytic granule polarization and degranulation. Intercellular adhesion molecule (ICAM)-1 on insect cells was sufficient to induce polarization of granules, but not degranulation, in resting NK cells. Conversely, engagement of the Fc receptor CD16 by rabbit IgG on insect cells induced degranulation without specific polarization. Lysis by resting NK cells occurred when polarization and degranulation were induced by the combined presence of ICAM-1 and IgG on insect cells. Engagement of receptor 2B4 by CD48 on insect cells induced weak polarization and no degranulation. However, coengagement of 2B4 and CD16 by their respective ligands resulted in granule polarization and cytotoxicity in the absence of leukocyte functional antigen-1–mediated adhesion to target cells. These data show that cytotoxicity by resting NK cells is controlled tightly by separate or cooperative signals from different receptors for granule polarization and degranulation.

Killer cell inhibitory receptors specific for HLA-C and HLA-B identified by direct binding and by functional transfer

Abstract An inhibitory signal is delivered to natural killer (NK) cells and a subset of cytotoxic T cells upon recognition of HLA class 1 molecules on target cells. We demonstrate that soluble forms of killer cell inhibitory receptors (KIR) bind directly and specifically to HLA-C alleles on transfected cells. Furthermore, transfer of individual KIR Into NK clones reconstituted recognition of HLA-C on target cells, leading to inhibition of lysis. Using such functional reconstitution, a related KIR that confers specificity for some HLA-B alleles was also identified. These KIR share conserved tyrosine phosphorylation motifs in their cytoplasmic tails. Thus, a single receptor in NK cells provides both specificity for HLA class I on target cells and the Inhibitory signal that prevents lysis.

Peptide specificity in the recognition of MHC class I by natural killer cell clones

Recognition by natural killer (NK) cells of major histocompatibility complex (MHC) class I molecules on target cells inhibits NK-mediated lysis. Here, inhibition of NK clones by HLA-B*2705 molecules mutated at single amino acids in the peptide binding site varied among HLA-B*2705-specific NK clones. In addition, a subset of such NK clones was inhibited by only one of several self peptides loaded onto HLA-B*2705 molecules expressed in peptide transporter-deficient cells, showing that recognition was peptide-specific. These data demonstrate that specific self peptides, complexed with MHC class I, provide protection from NK-mediated lysis.

Activation of NK cells by an endocytosed receptor for soluble HLA-G.

Signaling from endosomes is emerging as a mechanism by which selected receptors provide sustained signals distinct from those generated at the plasma membrane. The activity of natural killer (NK) cells, which are important effectors of innate immunity and regulators of adaptive immunity, is controlled primarily by receptors that are at the cell surface. Here we show that cytokine secretion by resting human NK cells is induced by soluble, but not solid-phase, antibodies to the killer cell immunoglobulin-like receptor (KIR) 2DL4, a receptor for human leukocyte antigen (HLA)-G. KIR2DL4 was constitutively internalized into Rab5-positive compartments via a dynamin-dependent process. Soluble HLA-G was endocytosed into KIR2DL4–containing compartments in NK cells and in 293T cells transfected with KIR2DL4. Chemokine secretion induced by KIR2DL4 transfection into 293T cells occurred only with recombinant forms of KIR2DL4 that trafficked to endosomes. The profile of genes up-regulated by KIR2DL4 engagement on resting NK cells revealed a proinflammatory/proangiogenic response. Soluble HLA-G induced secretion of a similar set of cytokines and chemokines. This unique stimulation of resting NK cells by soluble HLA-G, which is endocytosed by KIR2DL4, implies that NK cells may provide useful functions at sites of HLA-G expression, such as promotion of vascularization in maternal decidua during early pregnancy.