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.     

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.     

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.     

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.     

Human cytomegalovirus infection induces a rapid and sustained change in the expression of NK cell receptors on CD8+ T cells

The CD8(+) T cell compartment of human CMV-seropositive individuals characteristically contains a high proportion of cells that express NK cell receptors (NKRs) which may contribute to the surveillance of virus-infected cells. To test whether this enhanced expression is a direct and immediate result of CMV infection, we used DNA microarrays to analyze putative changes in the RNA expression level of 39 NKRs in CMV-specific CD8(+) T cells of renal transplant recipients experiencing primary CMV infection. Already in the acute phase of infection 29 NKRs were induced, of which 19 remained high 1 year after cessation of viral replication. Activating and inhibitory NKRs were induced to a similar extent. Detailed longitudinal flow cytometric analyses confirmed NKR changes at the protein level. Strikingly, a strong induction of CD94 on CD3(+) T cells was observed with surface expression of activating CD94(dim) NKG2C dimers appearing before inhibitory CD94(bright) NKG2A ones. After the acute phase of infection, the balance between inhibitory and activating receptors did not change. Thus, CMV infection induces a rapid and lasting change in the expression of NKRs on human CD8(+) T cells.     

CD1d-independent NKT cells in beta 2-microglobulin-deficient mice have hybrid phenotype and function of NK and T cells

Unlike CD1d-restricted NK1.1(+)TCRalphabeta(+) (NKT) cells, which have been extensively studied, little is known about CD1d-independent NKT cells. To characterize their functions, we analyzed NKT cells in beta(2)-microglobulin (beta(2)m)-deficient B6 mice. They are similar to NK cells and expressed NK cell receptors, including Ly49, CD94/NKG2, NKG2D, and 2B4. NKT cells were found in normal numbers in mice that are deficient in beta(2)m, MHC class II, or both. They were also found in the male HY Ag-specific TCR-transgenic mice independent of positive or negative selection in the thymus. For functional analysis of CD1d-independent NKT cells, we developed a culture system in which CD1d-independent NKT cells, but not NK, T, or most CD1d-restricted NKT cells, grew in the presence of an intermediate dose of IL-2. IL-2-activated CD1d-independent NKT cells were similar to IL-2-activated NK cells and efficiently killed the TAP-mutant murine T lymphoma line RMA-S, but not the parental RMA cells. They also killed beta(2)m-deficient Con A blasts, but not normal B6 Con A blasts, indicating that the cytotoxicity is inhibited by MHC class I on target cells. IL-2-activated NKT cells expressing transgenic TCR specific for the HY peptide presented by D(b) killed RMA-S, but not RMA, cells. They also killed RMA (H-2(b)) cells that were preincubated with the HY peptide. NKT cells from beta(2)m-deficient mice, upon CD3 cross-linking, secreted IFN-gamma and IL-2, but very little IL-4. Thus, CD1d-independent NKT cells are significantly different from CD1d-restricted NKT cells. They have hybrid phenotypes and functions of NK cells and T cells.     

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.     

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.     

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.     

KLRG1+NKG2A+ CD8 T cells mediate protection and participate in memory responses during γ-herpesvirus infection

Functional CD8 T cell effector and memory responses are generated and maintained during murine γ-herpesvirus 68 (γHV68) persistent infection despite continuous presentation of viral lytic Ags. However, the identity of the CD8 T cell subpopulations that mediate effective recall responses and that can participate in the generation of protective memory to a γ-herpesvirus infection remains unknown. During γHV68 persistence, ～75% of γHV68-specific CD8 T cells coexpress the NK receptors killer cell lectin-like receptor G1 (KLRG1) and NKG2A. In this study, we take advantage of this unique phenotype to analyze the capacity of CD8 T cells expressing or not expressing KLRG1 and NKG2A to mediate effector and memory responses. Our results show that γHV68-specific KLRG1(+)NKG2A(+) CD8 T cells have an effector memory phenotype as well as characteristics of polyfunctional effector cells such us IFN-γ and TNF-α production, killing capacity, and are more efficient at protecting against a γHV68 challenge than their NKG2A(-)KLRG1(-) counterparts. Nevertheless, γHV68-specific NKG2A(+)KLRG1(+) CD8 T cells express IL-7 and IL-15 receptors, can survive long-term without cognate Ag, and subsequently mount a protective response during antigenic recall. These results highlight the plasticity of the immune system to generate protective effector and proliferative memory responses during virus persistence from a pool of KLRG1(+)NKG2A(+) effector memory CD8 T cells.     

Human tumor-derived exosomes down-modulate NKG2D expression

NKG2D is an activating receptor for NK, NKT, CD8(+), and gammadelta(+) T cells, whose aberrant loss in cancer is a key mechanism of immune evasion. Soluble NKG2D ligands and growth factors, such as TGFbeta1 emanating from tumors, are mechanisms for down-regulating NKG2D expression. Cancers thereby impair the capacity of lymphocytes to recognize and destroy them. In this study, we show that exosomes derived from cancer cells express ligands for NKG2D and express TGFbeta1, and we investigate the impact of such exosomes on CD8(+) T and NK cell NKG2D expression and on NKG2D-dependent functions. Exosomes produced by various cancer cell lines in vitro, or isolated from pleural effusions of mesothelioma patients triggered down-regulation of surface NKG2D expression by NK cells and CD8(+) T cells. This decrease was rapid, sustained, and resulted from direct interactions between exosomes and NK cells or CD8(+) T cells. Other markers (CD4, CD8, CD56, CD16, CD94, or CD69) remained unchanged, indicating the selectivity and nonactivatory nature of the response. Exosomal NKG2D ligands were partially responsible for this effect, as down-modulation of NKG2D was slightly attenuated in the presence of MICA-specific Ab. In contrast, TGFbeta1-neutralizing Ab strongly abrogated NKG2D down-modulation, suggesting exosomally expressed TGFbeta as the principal mechanism. Lymphocyte effector function was impaired by pretreatment with tumor exosomes, as these cells exhibited poor NKG2D-dependent production of IFN-gamma and poor NKG2D-dependent killing function. This hyporesponsiveness was evident even in the presence of IL-15, a strong inducer of NKG2D. Our data show that NKG2D is a likely physiological target for exosome-mediated immune evasion in cancer.     

NK cells are effectors for resolvin E1 in the timely resolution of allergic airway inflammation

Immune responses are pathologically sustained in several common diseases, including asthma. To determine endogenous proresolving mechanisms for adaptive immune responses, we used a murine model of self-limited allergic airway inflammation. After cessation of allergen exposure, eosinophils and T cells were cleared concomitant with the appearance of increased numbers of NK cells in the lung and mediastinal lymph nodes. The mediastinal lymph node NK cells were activated, expressing CD27, CD11b, CD69, CD107a, and IFN-γ. NK cell depletion disrupted the endogenous resolution program, leading to delayed clearance of airway eosinophils and Ag-specific CD4(+) T cells. NK cell trafficking to inflamed tissues for resolution was dependent upon CXCR3 and CD62L. During resolution, eosinophils and Ag-specific CD4(+) T cells expressed NKG2D ligands, and a blocking Ab for the NKG2D receptor delayed clearance of these leukocytes. Of interest, NK cells expressed CMKLR1, a receptor for the proresolving mediator resolvin E1, and depletion of NK cells decreased resolvin E1-mediated resolution of allergic inflammation. Resolvin E1 regulated NK cell migration in vivo and NK cell cytotoxicity in vitro. Together, these findings indicate new functions in catabasis for NK cells that can also serve as targets for proresolving mediators in the resolution of adaptive immunity.     

Influence of In Vitro IL-2 or IL-15 Alone or in Combination with Hsp 70 Derived 14-Mer Peptide (TKD) on the Expression of NK Cell Activatory and Inhibitory Receptors on Peripheral Blood T Cells,B Cells and NKT Cells

Previous studies from Multhoff and colleagues reported that plasma membrane Hsp70 acts as a tumour-specific recognition structure for activated NK cells, and that the incubation of NK cells with Hsp70 and/or a 14-mer peptide derived from the N-terminal sequence of Hsp70 (TKDNNLLGRFELSG, TKD, aa 450–463) plus a low dose of IL-2 triggers NK cell proliferation and migration, and their capacity to kill cancer cells expressing membrane Hsp70. Herein, we have used flow cytometry to determine the influence of in vitro stimulation of peripheral blood mononuclear cells from healthy individuals with IL-2 or IL-15, either alone or in combination with TKD peptide on the cell surface expression of CD94, NK cell activatory receptors (CD16, NK2D, NKG2C, NKp30, NKp44, NKp46, NKp80, KIR2DL4, DNAM-1 and LAMP1) and NK cell inhibitory receptors (NKG2A, KIR2DL2/L3, LIR1/ILT-2 and NKR-P1A) by CD3+CD56+ (NKT), CD3+CD4+, CD3+CD8+ and CD19+ populations. NKG2D, DNAM-1, LAMP1 and NKR-P1A expression was upregulated after the stimulation with IL-2 or IL-15 alone or in combination with TKD in NKT, CD8+ T cells and B cells. CD94 was upregulated in NKT and CD8+ T cells. Concurrently, an increase in a number of CD8+ T cells expressing LIR1/ILT-2 and CD4+ T cells positive for NKR-P1A was observed. The proportion of CD8+ T cells that expressed NKG2D was higher after IL-2/TKD treatment, when compared with IL-2 treatment alone. In comparison with IL-15 alone, IL-15/TKD treatment increased the proportion of NKT cells that were positive for CD94, LAMP1 and NKRP-1A. The more potent effect of IL-15/TKD on cell surface expression of NKG2D, LIR1/ILT-2 and NKRP-1A was observed in B cells compared with IL-15 alone. However, this increase was not of statistical significance. IL-2/TKD induced significant upregulation of LAMP1 in CD8+ T cells compared with IL-2 alone. Besides NK cells, other immunocompetent cells present within the fraction of peripheral blood mononuclear cells were influenced by the treatment with low-dose interleukins themselves or in combination with hsp70 derived (TKD) peptide.     

Cutting edge: CD8+ T cell priming in the absence of NK cells leads to enhanced memory responses

It is uncertain whether NK cells modulate T cell memory differentiation. By using a genetic model that allows the selective depletion of NK cells, we show in this study that NK cells shape CD8(+) T cell fate by killing recently activated CD8(+) T cells in an NKG2D- and perforin-dependent manner. In the absence of NK cells, the differentiation of CD8(+) T cells is strongly biased toward a central memory T cell phenotype. Although, on a per-cell basis, memory CD8(+) T cells generated in the presence or the absence of NK cells have similar functional features and recall capabilities, NK cell deletion resulted in a significantly higher number of memory Ag-specific CD8(+) T cells, leading to more effective control of tumors carrying model Ags. The enhanced memory responses induced by the transient deletion of NK cells may provide a rational basis for the design of new vaccination strategies.     

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.     

NK cells enhance dendritic cell response against parasite antigens via NKG2D pathway

Recent studies have shown that NK-dendritic cell (DC) interaction plays an important role in the induction of immune response against tumors and certain viruses. Although the effect of this interaction is bidirectional, the mechanism or molecules involved in this cross-talk have not been identified. In this study, we report that coculture with NK cells causes several fold increase in IL-12 production by Toxoplasma gondii lysate Ag-pulsed DC. This interaction also leads to stronger priming of Ag-specific CD8+ T cell response by these cells. In vitro blockade of NKG2D, a molecule present on human and murine NK cells, neutralizes the NK cell-induced up-regulation of DC response. Moreover, treatment of infected animals with Ab to NKG2D receptor compromises the development of Ag-specific CD8+ T cell immunity and reduces their ability to clear parasites. These studies emphasize the critical role played by NKG2D in the NK-DC interaction, which apparently is important for the generation of robust CD8+ T cell immunity against intracellular pathogens. To the best of our knowledge, this is the first work that describes in vivo importance of NKG2D during natural infection.     

Transfer of T cell surface molecules to dendritic cells upon CD4+ T cell priming involves two distinct mechanisms

Activation of CD4(+) T cells by APCs occurs by multiple Ag recognition events including the exchange of costimulatory signals and cytokines. Additionally, the T cells acquire APC-derived surface molecules. Herein, we describe for the first time the transfer of human and murine T cell surface receptors to APCs after Ag-specific interaction. This transfer occurs in two qualitatively different phases. The first group of molecules (e.g., CD2) derived from the T cell surface was transferred rapidly after 2 h of interaction, was strongly bound on the DC surface (acid wash-resistant), was strictly dependent on dendritic cell-T cell contact, and transferred independently of T cell activation. The second group, including the CD3/TCR complex, CD27, and OX40, was of intracellular origin, transferred later after 10-16 h in a cell-cell contact-independent fashion, was noncovalently bound, and was strictly dependent on Ag-specific T cell activation. Functionally, murine dendritic cells that received TCR molecules from OVA-specific CD4(+) T cells after Ag-specific interaction were less efficient in priming naive CD4(+) T cells of the same specificity without losing their ability for CD8(+) T cell stimulation, indicating that the transferred TCR molecules mask the Ag-bearing MHC II molecules, thereby reducing their accessibility to following Ag-specific CD4(+) T cells. While the first group of transferred T cell surface molecules might facilitate the detachment of the CD4(+) T cell from the dendritic cell during the early scanning phases, the second group could play an important immunomodulatory role in intraclonal competition of T cells for APC access, making the physical presence of CD4(+) T cells unnecessary.