Expression of CD94/NKG2-A on human T lymphocytes is induced by IL-12: implications for adoptive immunotherapy

NK cell receptors (NKRs) are expressed on a subset of human T cells, predominantly CD8(+), within which they can modulate TCR-mediated functions. In an attempt to identify the mechanisms leading to NKR expression, we analyzed the capacity of IL-12 to modulate the expression by T cells of the components of the CD94/NKG2-A inhibitory receptor, a member of the C-type lectin-like family of NKR. We show that IL-12 induces the expression of NKG2-A and/or CD94 by CD8(+) T cells in culture, and that this induction was mediated neither by IFN-gamma nor by IL-15. We also show, using the redirected killing assay, that IL-12-induced expression of both CD94 and NKG2-A led to the acquisition by T cells of a functional inhibitory receptor. Expression of the CD94/NKG2-A inhibitory receptor was also induced by IL-12 during T cell Ag stimulation so that in the presence of this cytokine a high proportion of melanoma-reactive CTL induced from PBL by melanoma peptide stimulation expressed this receptor. This study emphasizes the implication of IL-12 in the modulation of immune responses through NKR induction.     

Viral and bacterial infections induce expression of multiple NK cell receptors in responding CD8(+) T cells

NK cells express several families of receptors that play central roles in target cell recognition. These NK cell receptors are also expressed by certain memory phenotype CD8(+) T cells, and in some cases are up-regulated in T cells responding to viral infection. To determine how the profile of NK receptor expression changes in murine CD8(+) T cells as they respond to intracellular pathogens, we used class I tetramer reagents to directly examine Ag-specific T cells during lymphocytic choriomeningitis virus and Listeria monocytogenes infections. We found that the majority of pathogen-specific CD8(+) T cells initiated expression of the inhibitory CD94/NKG2A heterodimer, the KLRG1 receptor, and a novel murine NK cell marker (10D7); conversely, very few Ag-specific T cells expressed Ly49 family members. The up-regulation of these receptors was independent of IL-15 and persisted long after clearance of the pathogen. The expression of CD94/NKG2A was rapidly initiated in naive CD8(+) T cells responding to peptide Ags in vitro and on many of the naive T cells that proliferate when transferred into lymphopenic (Rag-1(-/-)) hosts. Thus, CD94/NKG2A expression is a common consequence of CD8(+) T cell activation. Binding of the CD94/NKG2A receptor by its ligand (Qa-1(b)) did not significantly inhibit CD8(+) T cell effector functions. However, expression of CD94 and NKG2A transgenes partially inhibited early events of T cell activation. These subtle effects suggest that CD94/NKG2A-mediated inhibition of T cells may be limited to particular circumstances or may synergize with other receptors that are similarly up-regulated.     

Complex interplay of activating and inhibitory signals received by Vgamma9Vdelta2 T cells revealed by target cell beta2-microglobulin knockdown

Tumor cells often escape immunosurveillance by down-regulating MHC class I molecule expression. For human Vgamma9Vdelta2 T cells, a major peripheral blood T cell subset with broad antitumor reactivity, this down-regulation can affect signals transmitted by both the inhibitory and the activating MHC class I and Ib-specific NK receptors (NKRs) that these lymphocytes frequently express. To assess the overall impact of MHC down-regulation on Vgamma9Vdelta2 T cell activation, we used stable beta(2)-microglobulin knockdown to generate tumor cells with a approximately 10-fold down-modulation of all MHC class I molecules. This down-modulation had little effect on T cell proliferation or cytokine production, but modified tumor cell killing efficiency. Ab-blocking studies identified ILT2 as an important inhibitor of tumor cell killing by Vgamma9Vdelta2 T cells. Down-modulation of MHC class I and Ib molecules severely reduced ILT2 inhibitory signaling, but still allowed signaling by activating CD94-based receptors. It also unveiled a frequent enhancing effect of NKG2D on tumor killing by Vgamma9Vdelta2 T cells. Current models suggest that activating NKRs have less affinity for their MHC ligands than homologous inhibitory NKRs. Our results show that, despite this, activating NKRs recognizing MHC class I molecules play an important role in the increased killing by Vgamma9Vdelta2 T cells of tumor cells with down-regulated MHC class I molecule expression, and suggest that these T cells will best lyse tumor cells combining MHC class I molecule expression down-regulation with up-regulated NKG2D ligand expression.     

CD8 T cells expressing NK associated receptors are increased in melanoma patients and display an effector phenotype

CD8⁺ T cells can express NK-associated receptors (NKRs) that may regulate their cytolytic function. We have characterized the expression of several NKRs on peripheral blood CD8⁺ T cells from melanoma patients and compared them to age-matched healthy donors. The analysis performed includes HLA class I specific receptors (KIRs, LILRB1 and CD94/NKG2) and other NK receptors like CD57, CD56 and CD16. Melanoma patients showed a higher variability in the expression of NKRs on circulating CD8⁺ T cells than age-matched healthy donors. NKR expression on CD8⁺ T cells from melanoma patients showed a significant increase of KIR2DL2/L3/S2 (mAb gl183), CD244, CD57, CD56 and CD16. We have also found an increase of CD8⁺ CD28^– CD27^– T cells in melanoma patients. This subset represents terminally differentiated effector cells expressing CD244 and high levels of perforin. The expression of NKRs was also mainly restricted to this T cell subset. Altogether, circulating CD8⁺ T cells from melanoma patients display a distinct phenotype characterized by downregulation of costimulatory molecules and higher expression of NKRs. We suggest that the increased expression of NKRs on T cells may contribute to the final outcome of the immune response against melanoma both stimulating or inhibiting activation and differentiation to effector cells. Blocking inhibitory receptor function and enhancing activating receptors may represent new strategies with therapeutic potential against melanoma.     

CD94/NKG2A expression is associated with proliferative potential of CD8 T cells during persistent polyoma virus infection

Memory CD8 T cells comprise a critical component of durable immunity because of their capacity to rapidly proliferate and exert effector activity upon Ag rechallenge. During persistent viral infection, memory CD8 T cells repetitively encounter viral Ag and must maintain a delicate balance between limiting viral replication and minimizing immunopathology. In mice infected by polyoma virus, a natural mouse pathogen that establishes long-term persistent infection, the majority of persistence-phase antiviral CD8 T cells express the inhibitory NK cell receptor CD94/NKG2A. In this study, we asked whether CD94/NKG2A expression is associated with Ag-specific recall of polyoma virus-specific CD8 T cells. During the persistent phase of infection, polyoma virus-specific CD8 T cells that express CD94/NKG2A were found to preferentially proliferate; this proliferation was dependent on cognate Ag both in vitro and in vivo. In addition, CD94/NKG2A(+) polyoma-specific CD8 T cells have a markedly enhanced capacity to produce IL-2 upon ex vivo Ag stimulation compared with CD94/NKG2A(-) polyoma-specific CD8 T cells. Importantly, CD94/NKG2A(+) anti-polyoma virus CD8 T cells appear to be essential for Ag-specific recall responses in mice persistently infected by polyoma virus. Because of its higher proliferative potential and capacity to produce IL-2, we propose that the CD94/NKG2A(+) subpopulation represents a less differentiated state than the CD94/NKG2A(-) subpopulation. Identification of proliferation-competent subpopulations of memory CD8 T cells should prove valuable in designing therapeutic vaccination strategies for persistent viral infections.     

Expression of inhibitory receptors Ly49E and CD94/NKG2 on fetal thymic and adult epidermal TCR V gamma 3 lymphocytes

Ly49 and CD94/NKG2 inhibitory receptors are predominantly expressed on murine NK cells, but they are also expressed on a subpopulation of peripheral CD8 memory TCR alphabeta lymphocytes. In this study we demonstrate that Ly49E and CD94/NKG2 receptors are expressed on mature TCR Vgamma3(+) cells in the fetal thymus. Expression correlated with a memory phenotype, such as expression of CD44, 2B4, and IL-2Rbeta (CD122), and absence of IL-2Ralpha (CD25) expression. No expression of Ly49A, C, D, G2, or I receptors was observed. This phenotype is similar to that of fetal thymic NK cells. Skin-located Vgamma3 T cells, the progeny of fetal thymic Vgamma3 cells, also expressed CD94/NKG2 and Ly49E but not the other members of the Ly49 family. The development and survival of Ly49E(+) or CD94/NKG2(+) Vgamma3 T lymphocytes was not dependent upon expression of MHC class I molecules. The cytotoxicity of TCR Vgamma3 cells was inhibited when Qdm, the ligand for CD94/NKG2, was presented by Qa1(b)-transfected target cells. Also, upon cross-linking of CD94/NKG2 with mAb 3S9, TCR Vgamma3 thymocytes were prevented from killing FcgammaR(+) P815 target cells. These effects were most pronounced in the CD94/NKG2(high) subpopulation as compared with the CD94/NKG2(low) subpopulation of Vgamma3 cells. Our data demonstrate that Vgamma3 T cells expressing inhibitory Ly49E and CD94/NKG2 receptors are mature and display a memory phenotype, and that CD94/NKG2 functions as an inhibitory receptor on these T lymphocytes.     

Phenotypic and functional changes of cytotoxic CD56pos natural T cells determine outcome of acute hepatitis C virus infection

Innate CD56(pos) natural killer (NK) and natural T (NT) cells comprise important hepatic antiviral effector lymphocytes whose activity is fine-tuned through surface NK receptors (NKRs). Dysregulation of NKRs in patients with long-standing hepatitis C virus (HCV) infection has been shown, but little is known regarding NKRs in acute infection. Treatment-na?ve patients with acute HCV (n = 22), including 10 with spontaneous recovery, were prospectively studied. CD56(pos) NT levels were reduced early in acute HCV infection and did not fluctuate over time. In resolving HCV infection, NT cells with a more activated phenotype (lower CD158A and higher natural cytotoxicity receptor expression) at baseline predated spontaneous recovery. Moreover, NKG2A expression on CD56(+) NT cells correlated directly with circulating HCV RNA levels. Deficient interleukin-13 (IL-13) production by NT cells and reduced IL-2-activated killing (LAK) at baseline were associated with the ultimate development of persistence. These results indicate a previously unappreciated role for NT cells in acute HCV infection and identify a potential target for pharmacologic manipulation.     

Lymphocyte activation in response to melanoma: interaction of NK-associated receptors and their ligands

In recent years, studies on the molecular and cellular mechanisms of immune responses against melanoma have contributed to a better understanding of how these tumours can be recognised by cytotoxic cells and the mechanisms they have developed to escape from innate and adaptive immunity. Lysis of melanoma cells by natural killer (NK) cells and cytolytic T cells is the result of a fine balance between signals transmitted by activating and inhibitory receptors. In addition to the T cell receptor, these were initially described as NK cell-associated receptors (NKRs) and were later also found on subsets of T lymphocytes, particularly effector-memory and terminally differentiated CD8 T cells. An increase of NKR(+)CD8(+) T cells has been found in melanoma patients, correlating with the expansion of differentiated effector CD8(+)CD28(null) CD27(null) T cells. NKRs can regulate the lysis of target cells expressing appropriate ligands. Activating receptors recognise ligands on tumours whereas inhibitory receptors are specific for MHC class I antigens and sense missing self. Altered expression of MHC class I antigens is frequently found on melanoma cells, preventing recognition by specific cytolytic T cells but favouring NK cell recognition. Changes in the expression of NKR-ligands in melanoma contribute in explaining the differences in the capacity of cytotoxic immune cells to control melanoma growth and dissemination.     

Alteration of inhibitory and activating NK cell receptor expression on NK cells in HIV-infected Chinese

Natural killer (NK) cell function, based on the expression of activating and inhibitory natural killer receptors (NKRs), may become abnormal during human immunodeficiency virus (HIV) infection. In this study, we investigated changes in receptor expression with individual and combinational analysis on NK cell subsets in HIV-infected Chinese. The results showed that natural killer group 2 member D (NKG2D) expression on total NK cells decreased significantly in HIV infection, while the expressions of natural killer group 2 member A (NKG2A) and killer cell immunoglobulin-like receptor, three domains, long cytoplasmic tail 1 (KIR3DL1) on total NK cells were not significantly different between any of the groups including HIV-positive treatment-naïve group, AIDS treatment-naïve group, HAART-treatment AIDS group and HIV-negative control group. Individual analysis of NKG2A⁺ and KIR3DL1⁺ cells revealed no significant differences in expression in any NK cell subsets between any of the groups, but the combinational analysis of NKG2D⁻NKG2A⁺, and NKG2D⁻KIR3DL1⁺ on the NK CD56^dim cell subset in the AIDS group were increased compared to the HIV-negative control group. On the contrary, NKG2D⁻NKG2A⁺ expression on the CD56^bright subset decreased in the AIDS group compared to the control group. Highly active antiretroviral therapy (HAART) treatment almost completely restored the levels of these receptor expressions. The results indicate that the distinct alteration of activating and inhibitory NKR expression on NK cells and its subsets occurred during HIV progression. Moreover, the imbalanced change of activating and inhibitory NKRs on NK cells and its subsets may explain the impaired NK cell immunity in HIV infected individuals.     

Development and function of CD94-deficient natural killer cells

The CD94 transmembrane-anchored glycoprotein forms disulfide-bonded heterodimers with the NKG2A subunit to form an inhibitory receptor or with the NKG2C or NKG2E subunits to assemble a receptor complex with activating DAP12 signaling proteins. CD94 receptors expressed on human and mouse NK cells and T cells have been proposed to be important in NK cell tolerance to self, play an important role in NK cell development, and contribute to NK cell-mediated immunity to certain infections including human cytomegalovirus. We generated a gene-targeted CD94-deficient mouse to understand the role of CD94 receptors in NK cell biology. CD94-deficient NK cells develop normally and efficiently kill NK cell-susceptible targets. Lack of these CD94 receptors does not alter control of mouse cytomegalovirus, lymphocytic choriomeningitis virus, vaccinia virus, or Listeria monocytogenes. Thus, the expression of CD94 and its associated NKG2A, NKG2C, and NKG2E subunits is dispensable for NK cell development, education, and many NK cell functions.     

The repertoire of killer cell Ig-like receptor and CD94:NKG2A receptors in T cells: clones sharing identical alpha beta TCR rearrangement express highly diverse killer cell Ig-like receptor patterns

Killer cell Ig-like receptor (KIR) and CD94:NKG2A molecules were first defined as human NK cell receptors (NKR), but now are known to be expressed and to function on subpopulations of T cells. Here the repertoires of KIR and CD94:NKG2A expression by T cells from two donors were examined and compared with their previously defined NK cell repertoires. T cell clones generated from peripheral blood of both donors expressed multiple NKR in different combinations and used the range of receptors expressed by NK cells. In both donors alpha beta T cells less frequently expressed the inhibitory receptors CD94:NKG2A and KIR2DL1 than either gamma delta T cells or NK cells. In contrast to NK cells, not all NKR(+) T cells expressed an inhibitory receptor for autologous HLA class I. This lack of specific inhibitory NKR was especially apparent on alpha beta T cells of one donor. Overall, alpha beta T cells exhibited a distinct pattern of NKR expression different from that of gamma delta T and NK cells, which expressed highly similar NKR repertoires. In one donor, analysis of TCR rearrangement revealed a dominant subset of NKR(+) T cells sharing identical TCR alpha- and beta-chains. Remarkably, among 55 T cell clones sharing the same TCR alpha beta rearrangement 18 different KIR phenotypes were seen, suggesting that KIR expression was initiated subsequently to TCR rearrangement.     

Coordinated expression of Ig-like inhibitory MHC class I receptors and acquisition of cytotoxic function in human CD8+ T cells

MHC class I-specific inhibitory receptors are expressed by a subset of memory-phenotype CD8(+) T cells. Similar to NK cells, MHC class I-specific inhibitory receptors might subserve on T cells an important negative control that participates to the prevention of autologous damage. We analyzed here human CD8(+) T cells that express the Ig-like MHC class I-specific inhibitory receptors: killer cell Ig-like receptor (KIR) and CD85j. The cell surface expression of Ig-like inhibitory MHC class I receptors was found to correlate with an advanced stage of CD8(+) T cell maturation as evidenced by the reduced proliferative potential of KIR(+) and CD85j(+) T cells associated with their high intracytoplasmic perforin content. This concomitant regulation might represent a safety mechanism to control potentially harmful cytolytic CD8(+) T cells, by raising their activation threshold. Yet, KIR(+) and CD85j(+) T cells present distinct features. KIR(+)CD8(+) T cells are poor IFN-gamma producers upon TCR engagement. In addition, KIR are barely detectable at the surface of virus-specific T cells during the course of CMV or HIV-1 infection. By contrast, CD85j(+)CD8(+) T cells produce IFN-gamma upon TCR triggering, and represent a large fraction of virus-specific T cells. Thus, the cell surface expression of Ig-like inhibitory MHC class I receptors is associated with T cell engagement into various stages of the cytolytic differentiation pathway, and the cell surface expression of CD85j or KIR witnesses to the history of qualitatively and/or quantitatively distinct T cell activation events.     

Preferential survival of CD8 T and NK cells expressing high levels of CD94

The Qa-1(b)/Qdm tetramer binds to CD94/NKG2 receptors expressed at high levels on approximately 50% of murine NK cells. Although very few CD8 T cells from naive mice express CD94/NKG2 receptors, approximately 50% of CD8 T cells taken from mice undergoing a secondary response against Listeria monocytogenes (LM) are CD94(high) and bind the tetramer. Although CD94(int) NK cells do not bind the tetramer, CD94(int) CD8 T cells do, and this binding is dependent on the CD8 coreceptor. We found that the extent of apoptosis in CD8 T and NK cells was inversely related to the expression of CD94, with lower levels of apoptosis seen in CD94(high) cells after 1-3 days of culture. The difference in CD8 T cell survival was evident as early as 6 h after culture and persisted until nearly all the CD94(neg/int) cells were apoptotic by 48 h. In contrast, expression of inhibitory Ly-49A,G2,C/I molecules was associated with higher levels of apoptosis. Cross-linking CD94/NKG2 receptors on CD8 T cells from a mouse undergoing an LM infection further reduced the percentage of apoptotic cells on the CD94-expressing populations, while cross-linking Ly-49I had no effect on CD8 T cells expressing Ly-49I. Cross-linking CD3 on CD8 T cells from a mouse undergoing a secondary LM infection increases the extent of apoptosis, but this is prevented by cross-linking CD94/NKG2 receptors at the same time. Similar results were observed with NK cells in that the CD94(high) population displayed less apoptosis than CD94(int) cells after 1-3 days in culture. Therefore, the expression of CD94/NKG2 is correlated with a lower level of apoptosis and may play an important role in the maintenance of CD8 T and NK cells.     

IL-2-activated CD8+CD44high cells express both adaptive and innate immune system receptors and demonstrate specificity for syngeneic tumor cells

CD8(+) T cells depend on the alphabeta TCR for Ag recognition and function. However, Ag-activated CD8(+) T cells can also express receptors of the innate immune system. In this study, we examined the expression of NK receptors on a population of CD8(+) T cells expressing high levels of CD44 (CD8(+)CD44(high) cells) from normal mice. These cells are distinct from conventional memory CD8(+) T cells and they proliferate and become activated in response to IL 2 via a CD48/CD2-dependent mechanism. Before activation, they express low or undetectable levels of NK receptors but upon activation with IL-2 they expressed significant levels of activating NK receptors including 2B4 and NKG2D. Interestingly, the IL-2-activated cells demonstrate a preference in the killing of syngeneic tumor cells. This killing of syngeneic tumor cells was greatly enhanced by the expression of the NKG2D ligand Rae-1 on the target cell. In contrast to conventional CD8(+) T cells, IL-2-activated CD8(+)CD44(high) cells express DAP12, an adaptor molecule that is normally expressed in activated NK cells. These observations indicate that activated CD8(+)CD44(high) cells express receptors of both the adaptive and innate immune system and may play a unique role in the surveillance of host cells that have been altered by infection or transformation.     

Cutting Edge: Distinct NK receptor profiles are imprinted on CD8 T cells in the mucosa and periphery during the same antigen challenge: role of tissue-specific factors

NK cell receptors (NKRs) modulate T lymphocyte responses by modifying the Ag activation threshold. However, what governs their expression on T cells remains unclear. In this study we show that different NKRs are imprinted on CD8 T cells in the gut mucosa and periphery during the same Ag challenge. After a viral, bacterial, and tumor challenge, most CD8 peritoneal exudate lymphocytes expressed NKG2A but not 2B4. In contrast, most CD8 intraepithelial lymphocytes exhibited 2B4 but not NKG2A. Our data suggest that tissue-specific factors may determine the pattern of NKR expression. In the gut, CD70 licensing appears to promote 2B4 induction on mucosal CD8 T cells. Conversely, retinoic acid produced by the intestinal dendritic cells may suppress NKG2A expression. Thus, tissue-specific factors regulate NKR expression and may confer T cells with differing effector functions in a tissue and site-specific manner.     

Blockade of NKG2D signaling prevents the development of murine CD4+ T cell-mediated colitis

It has been recently demonstrated that NKG2D is an activating costimulatory receptor on natural killer (NK) cells, natural killer T (NKT) cells, activated CD8(+) T cells, and gammadelta T cells, which respond to cellular stress, such as inflammation, transformation, and infection. Here we show that intestinal inflammation in colitic SCID mice induced by adoptive transfer of CD4(+)CD45RB(high) T cells is characterized by significant increase of CD4(+)NKG2D(+) T cells and constitutive expression of NKG2D ligands, such as H60, Mult-1, and Rae-1, by lamina propria CD11c(+) dendritic cells. Furthermore, treatment with nondepleting and neutralizing anti-NKG2D MAb after transfer of CD4(+)CD45RB(high) T cells into SCID mice significantly suppressed wasting disease with colitis, abrogated leukocyte infiltration, and reduced production of IFN-gamma by lamina propria CD4(+) T cells. These findings demonstrate that NKG2D signaling pathway is critically involved in CD4(+) T cell-mediated disease progression and suggest a new therapeutic target for inflammatory bowel diseases.     

Toxoplasma gondii Infection Regulates the Balance of Activating and Inhibitory Receptors on Decidual Natural Killer Cells

Inhibitory receptors and activating receptor expressed on decidual natural killer (dNK) cells are generally believed to be important in abnormal pregnancy outcomes and induced adverse pregnancy. However, if Toxoplasma gondii (T. gondii) infection induced abnormal pregnancy was related to dNK cells changes is not clear. In this study, we used human dNK cells co-cultured with human extravillous cytotrophoblast (EVT) cells following YFP-Toxoplasma gondii (YFP-T. gondii) infection in vitro and established animal pregnant infection model. Levels of inhibitory receptors KIR2DL4 and ILT-2, their ligand HLA-G, and activating receptor NKG2D in human decidua, and NKG2A and its ligand Qa-1 and NKG2D in mice uterine were analyzed by real-time PCR and flow cytometry with levels of NKG2D significantly higher than those of KIR2DL4 and ILT-2 in vitro and in invo. The level of NKG2D was positively correlated with cytotoxic activity of dNK cells in vitro. Numbers of abnormal pregnancies were significantly greater in the infected group than in the control group. This result demonstrated that the increased NKG2D expression and imbalance between inhibitory receptors of dNK cells and HLA-G may contribute to abnormal pregnancy outcomes observed upon maternal infection with T. gondii.     

Human leukocyte antigen E in human cytomegalovirus infection: friend or foe?

Human cytomegalovirus (HCMV) is a well-studied β-herpesvirus virus, which adopts a variety of strategies to evade immune surveillance. It has been reported that in HCMV-infected cells, classical major histocompatibility (MHC) class I molecules are down-regulated, but the MHC class Ib molecule human leukocyte antigen (HLA)-E is normally expressed or even overexpressed on the cell surface. HLA-E has been first described to interact with CD94/NKG2 receptors expressed mainly on the surface of natural killer (NK) cells, thus confining its role to the regulation of NK-cell function. The engagement of CD94/NKG2A with HLA-E, with a signal peptide of the HCMV glycoprotein UL40, usually induces inhibitory signals. However, HLA-E also serves as a ligand for the TCR expressed by αβCD8(+) T cells. Recognition of peptides presented by HLA-E may result in CD8(+) effector T-cell activation. These findings will help to understand more on both pathogenic and protective roles of HLA-E in HCMV infection. In this review, we discussed recent studies about the roles of HLA-E in HCMV infection.     

NK cell CD94/NKG2A inhibitory receptors are internalized and recycle independently of inhibitory signaling processes

Human CD94/NKG2A is an inhibitory receptor that recognizes HLA-E and is expressed by NK cells and a subset of T cells. We have analyzed the cellular trafficking of the CD94/NKG2A receptor using the NKL cell line and peripheral blood NK cells. Flow cytometric, confocal microscopic, and biochemical analyses show that CD94/NKG2A continuously recycles in an active process that requires the cytoskeleton between the cell surface and intracellular compartments that are distinguishable from recycling compartments used by well-characterized receptors, such as transferrin receptor (CD71). CD94/NKG2A, an inhibitory receptor, traffics differently from the closely related CD94/NKG2C molecule, an activating receptor. Using transfection/expression analyses of wild-type and mutant CD94/NKG2A molecules in the HLA-E negative rat basophilic cell line RBL-2H3, we demonstrate that CD94/NKG2A internalization is independent of ligand cross-linking or the presence of functional immunoreceptor tyrosine-based inhibition motifs. Thus, the mechanisms that control cell surface homeostasis of CD94/NKG2A are independent of functional signaling.