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.     

Orderly and nonstochastic acquisition of CD94/NKG2 receptors by developing NK cells derived from embryonic stem cells in vitro

In mice there are two families of MHC class I-specific receptors, namely the Ly49 and CD94/NKG2 receptors. The latter receptors recognize the nonclassical MHC class I Qa-1(b) and are thought to be responsible for the recognition of missing-self and the maintenance of self-tolerance of fetal and neonatal NK cells that do not express Ly49. Currently, how NK cells acquire individual CD94/NKG2 receptors during their development is not known. In this study, we have established a multistep culture method to induce differentiation of embryonic stem (ES) cells into the NK cell lineage and examined the acquisition of CD94/NKG2 by NK cells as they differentiate from ES cells in vitro. ES-derived NK (ES-NK) cells express NK cell-associated proteins and they kill certain tumor cell lines as well as MHC class I-deficient lymphoblasts. They express CD94/NKG2 heterodimers, but not Ly49 molecules, and their cytotoxicity is inhibited by Qa-1(b) on target cells. Using RT-PCR analysis, we also report that the acquisition of these individual receptor gene expressions during different stages of differentiation from ES cells to NK cells follows a predetermined order, with their order of acquisition being first CD94; subsequently NKG2D, NKG2A, and NKG2E; and finally, NKG2C. Single-cell RT-PCR showed coexpression of CD94 and NKG2 genes in most ES-NK cells, and flow cytometric analysis also detected CD94/NKG2 on most ES-NK cells, suggesting that the acquisition of these receptors by ES-NK cells in vitro is nonstochastic, orderly, and cumulative.     

Diversity of NK cell receptor repertoire in adult and neonatal mice.

Murine NK cytotoxicity is regulated by two families of MHC class I-specific receptors, namely Ly49 and CD94/NKG2. We developed a single-cell RT-PCR method to analyze expression of all known Ly49 and NKG2A genes in individual NK cells and determined the receptor repertoires of NK cells from adult and neonatal (1-wk-old) C57BL/6 mice. In adult mouse NK cells, up to six different receptors were coexpressed in random combinations. Of 62 NK cells examined, 42 different patterns of receptor expression were observed. Most of them expressed at least one Ly49, whereas NKG2A was detected in 32% of the cells. Over 75% of them expressed Ly49C, I, or NKG2A, which are thought to recognize self-class I MHC (H-2b). Coexpression of multiple Ly49 receptors and NKG2A was stochastic. In contrast, very few neonatal NK cells expressed any Ly49, but almost 60% of them expressed NKG2A. These results demonstrate that adult NK cells are quite heterogeneous and have diverse receptor repertoires. They also suggest that the expression of NKG2A precedes Ly49 expression in NK cell ontogeny, and NKG2A is a major inhibitory receptor in neonatal NK cells.     

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.     

Mouse CD94 participates in Qa-1-mediated self recognition by NK cells and delivers inhibitory signals independent of Ly-49

Inhibitory receptors expressed on NK cells recognize MHC class I molecules and transduce negative signals to prevent the lysis of healthy autologous cells. The lectin-like CD94/NKG2 heterodimer has been studied extensively as a human inhibitory receptor. In contrast, in mice, another lectin-like receptor, Ly-49, was the only known inhibitory receptor until the recent discovery of CD94/NKG2 homologues in mice. Here we describe the expression and function of mouse CD94 analyzed by a newly established mAb. CD94 was detected on essentially all NK and NK T cells as well as small fractions of T cells in all mouse strains tested. Two distinct populations were identified among NK and NK T cells, CD94(bright) and CD94(dull) cells, independent of Ly-49 expression. The anti-CD94 mAb completely abrogated the inhibition of target killing mediated by NK recognition of Qa-1/Qdm peptide on target cells. Importantly, CD94(bright) but not CD94(dull) cells were found to be functional in the Qa-1/Qdm-mediated inhibition. In the presence of the mAb, activated NK cells showed substantial cytotoxicity against autologous target cells as well as enhanced cytotoxicity against allogeneic and "missing self" target cells. These results suggest that mouse CD94 participates in the protection of self cells from NK cytotoxicity through the Qa-1 recognition, independent of inhibitory receptors for classical MHC class I such as Ly-49.     

Regulation of NKT cells by Ly49: analysis of primary NKT cells and generation of NKT cell line

TCRalphabeta(+)NK1.1(+) (NKT) cells are known to express various NK cell-associated molecules including the Ly49 family of receptors for MHC class I, but its functional significance has been unclear. Here, we examined the expression of Ly49A, C/I and G2 on various NKT cell populations from normal and MHC class I-deficient C57BL/6 mice as well as their responsiveness to alpha-galactosylceramide (alpha-GalCer), a potent stimulator of CD1d-restricted NKT cells. The frequency and the level of Ly49 expression varied among NKT cells from different tissues, and were regulated by the expression of MHC class I and CD1d in the host. Stimulation of various NKT cells with alpha-GalCer suggested that Ly49 expression inversely correlates with the responsiveness of NKT cells to alpha-GalCer. Moreover, alpha-GalCer presented by normal dendritic cells stimulated purified Ly49(-), but not Ly49(+), splenic NKT cells, whereas MHC class I-deficient dendritic cells presented alpha-GalCer to both Ly49(+) and Ly49(-) NKT cells equally well. Therefore, MHC class I on APCs seems to inhibit activation of NKT cells expressing Ly49. To further characterize CD1d-restricted NKT cells, we generated an alpha-GalCer-responsive NKT cell line from thymocytes. The line could only be generated from Ly49(-)NK1.1(+)CD4(+) thymocytes but not from other NKT cell subsets, and it lost expression of NK1.1 and CD4 during culture. Together, these results indicate the functional significance of Ly49 expression on NKT cells.     

A "chimeric" C57l-derived Ly49 inhibitory receptor resembling the Ly49D activation receptor.

Ly49D is a natural killer (NK) cell activation receptor that is responsible for differential mouse inbred strain-determined lysis of Chinese hamster ovary (CHO) cells. Whereas C57BL/6 NK cells kill CHO, BALB/c-derived NK cells cannot kill because they lack expression of Ly49D. Furthermore, the expression of Ly49D, as detected by monoclonal antibody 4E4, correlates well with CHO lysis by NK cells from different inbred strains. However, one discordant mouse strain was identified; C57L NK cells express the mAb 4E4 epitope but fail to lyse CHO cells. Herein we describe a Ly49 molecule isolated from C57L mice that is recognized by mAb 4E4 (anti-Ly49D). Interestingly, this molecule shares extensive similarity to Ly49D(B6) in its extracellular domain, but its cytoplasmic and transmembrane domains are identical to the inhibitory receptor Ly49A(B6), including a cytoplasmic ITIM. This molecule bears substantial overall homology to the previously cloned Ly49O molecule from 129 mice the serologic reactivity and function of which were undefined. Cytotoxicity experiments revealed that 4E4(+) LAK cells from C57L mice failed to lyse CHO cells and inhibited NK cell function in redirected inhibition assays. MHC class I tetramer staining revealed that the Ly49O(C57L)-bound H-2D(d) and lysis by 4E4(+) C57L LAK cells is inhibited by target H-2D(d). The structural basis for ligand binding was also examined in the context of the recent crystallization of a Ly49A-H-2D(d) complex. Therefore, this apparently "chimeric" Ly49 molecule serologically resembles an NK cell activation receptor but functions as an inhibitory receptor.     

Cutting edge: expression of functional CD94/NKG2A inhibitory receptors on fetal NK1.1+Ly-49- cells: a possible mechanism of tolerance during NK cell development.

Fetal liver- and thymus-derived NK1.1+ cells do not express known Ly-49 receptors. Despite the absence of Ly-49 inhibitory receptors, fetal and neonatal NK1.1+Ly-49- cells can distinguish between class Ihigh and class Ilow target cells, suggesting the existence of other class I-specific inhibitory receptors. We demonstrate that fetal NK1. 1+Ly-49- cell lysates contain CD94 protein and that a significant proportion of fetal NK cells are bound by Qa1b tetramers. Fetal and adult NK cells efficiently lyse lymphoblasts from Kb-/-Db-/- mice. Qa1b-specific peptides Qdm and HLA-CW4 leader peptide specifically inhibited the lysis of these blasts by adult and fetal NK cells. Qdm peptide also inhibited the lysis of Qa1b-transfected human 721.221 cells by fetal NK cells. Taken together, these results suggest that the CD94/NKG2A receptor complex is the major known inhibitory receptor for class I (Qa1b) molecules on developing fetal NK cells.     

Stochastic acquisition of Qa1 receptors during the development of fetal NK cells in vitro accounts in part but not in whole for the ability of these cells to distinguish between class I-sufficient and class I-deficient targets.

Fetal mouse NK cells are grossly deficient in the expression of Ly49 molecules yet show a limited ability to distinguish between wild-type and MHC class I-deficient target cells. In this paper we report that during their development in vitro from immature thymic progenitors, a proportion of C57BL/6 fetal NK cells acquires receptors for a soluble form of the nonclassical class I molecule Qa1b associated with the Qdm peptide, but not for soluble forms of the classical class I molecules Kb and Db. The acquisition of these Qa1 receptors occurs in a stochastic manner that is strictly controlled by cytokines, and in particular is strongly inhibited by IL-4. All fetal NK clones tested, including those that lack detectable Qa1 receptors, express mRNA for CD94 and for both inhibitory and noninhibitory members of the NKG2 family. Fetal NK cells lacking receptors for Qa1 (and also for classical class I molecules) cannot distinguish between wild-type and class I-deficient blasts but, surprisingly, distinguish efficiently between certain wild-type and class I-deficient tumor cells. A variant line that lacks several members of the NKG2 family kills both types of tumor cell equally well, suggesting the existence of NKG2-containing inhibitory receptors that recognize as yet undefined nonclassical class I molecules of restricted distribution.     

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.     

Ly49I NK cell receptor transgene inhibition of rejection of H2b mouse bone marrow transplants

The Ly49 family of genes encode NK cell receptors that bind class I MHC Ags and transmit negative signals if the cytoplasmic domains have immunoregulatory tyrosine-based inhibitory motifs (ITIMs). 5E6 mAbs recognize Ly49C and Ly49I receptors and depletion of 5E6+ NK cells prevents rejection of allogeneic or parental-strain H2d bone marrow cell (BMC) grafts. To determine the function of the Ly49I gene in the rejection of BMC grafts, we transfected fertilized eggs of FVB mice with a vector containing DNA for B6 strain Ly49I (Ly49IB6). Ly49IB6 is ITIM+ and is recognized by 5E6 as well as Ly49I-specific 8H7 mAbs. Normal FVB H2q mice reject H2b but not H2d BMC allografts, and the rejection of H2b BMC was inhibited partially by anti-NK1.1 and completely by anti-asialo GM1, but not by anti-CD8, Abs. In FVB mice, NK1.1 is expressed on only 60% NK cells. FVB. Ly49IB6 hosts failed to reject H2d or H2b BMC, but did reject class I-deficient TAP-1-/- BMC, indicating that NK cells were functional. Nondepleting doses of anti-Ly49I Abs reversed the acceptance of H2b BMC by FVB.Ly49IB6 mice. FVB.Ly49IB6+/- mice were crossed and back-crossed with 129 mice-H2b, 5E6-, poor responders to H2d BMC grafts. While transgene-negative H2b/q F1 or first-generation back-crossed mice rejected H2b marrow grafts (hybrid resistance), transgene-positive mice did not. Thus B6 strain Ly49I receptors transmit inhibitory signals from H2b MHC class I molecules. Moreover, Ly49IB6 has no positive influence on the rejection of H2d allografts.     

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.     

Costimulation of multiple NK cell activation receptors by NKG2D

The activation of NK cells is mediated through specific interactions between activation receptors and their respective ligands. Little is known, however, about whether costimulation, which has been well characterized for T cell activation, occurs in NK cells. To study the function of NKG2D, a potential NK costimulatory receptor, we have generated two novel hamster mAbs that recognize mouse NKG2D. FACS analyses demonstrate that mouse NKG2D is expressed on all C57BL/6 IL-2-activated NK (lymphokine-activated killer (LAK)) cells, all splenic and liver NK cells, and approximately 50% of splenic NKT cells. Consistent with limited polymorphism of NKG2D, its sequence is highly conserved, and the anti-NKG2D mAbs react with NK cells from a large number of different mouse strains. In chromium release assays, we show that stimulation of NK cells with anti-NKG2D mAb can redirect lysis. Also, enhanced lysis of transfected tumor targets expressing NKG2D ligand could be inhibited by addition of anti-NKG2D mAb. Interestingly, stimulation of LAK cells via NKG2D alone does not lead to cytokine release. However, stimulation of LAK via both an NK activation receptor (e.g., CD16, NK1.1, or Ly-49D) and NKG2D leads to augmentation of cytokine release compared with stimulation through the activation receptor alone. These results demonstrate that NKG2D has the ability to costimulate multiple NK activation receptors.     

Cutting edge: the mouse NK cell-associated antigen recognized by DX5 monoclonal antibody is CD49b (alpha 2 integrin, very late antigen-2)

DX5 mAb is a useful reagent because it stains NK cells from all mouse strains examined. We have identified the molecule recognized by DX5 mAb by using a retrovirus-mediated expression cloning system. A 5-kb cDNA encoding a protein that is reactive with the DX5 mAb was isolated from a NK cell cDNA library, and this molecule was identical with CD49b (very late Ag-2, alpha(2) integrin). The DX5 mAb reacted with transfectants expressing CD49b, and binding of DX5 to the NK cells and CD49b transfectants was blocked in the presence of other anti-CD49b mAbs. When NK1.1(+) NK cells were cultured with IL-2, they progressively lost reactivity with DX5 mAb as a consequence of cellular proliferation. Cytotoxicity mediated by the DX5(+) NK cells was dramatically higher as compared with DX5(-) NK cells. Therefore, DX5 mAb recognizes CD49b and can be used to define functionally distinct subsets of NK cells.     

Inducible expression of the gp49B inhibitory receptor on NK cells

Murine NK cells express inhibitory receptors belonging to the C-type lectin-like (Ly-49, CD94/NKG2) and Ig superfamily-related (gp49) receptors. The murine gp49B receptor displays structural homology with human killer inhibitory receptors, and was previously identified to be a receptor on mast cells and activated NK cells. The gp49B receptor is highly related to gp49A, a receptor with unknown function. In this study, using a novel mAb produced against soluble gp49B molecules that cross-reacts with gp49A, we examined the cellular distribution and function of these receptors. gp49 is constitutively expressed on cells of the myeloid lineage throughout development, as well as on mature cells. Importantly, gp49 is not expressed on spleen- and liver-derived lymphocytes, including NK cells, but its expression is induced in vitro on NK cells following IL-2 stimulation, or in vivo by infection with murine CMV. Molecular studies revealed that both the immunoreceptor tyrosine-based inhibitory motif-containing gp49B as well as immunoreceptor tyrosine-based inhibitory motif-less gp49A receptors are up-regulated on NK cells following murine CMV infection. When co-cross-linked with NK1.1, gp49B can inhibit NK1.1-mediated cytokine release by NK cells. Taken together, these studies demonstrate that the expression of gp49B on NK cells is regulated, providing the first example of an in vivo activation-induced NK cell inhibitory receptor, in contrast to the constitutively expressed Ly49 family.     

Natural Killer Cell Tolerance Persists Despite Significant Reduction of Self MHC Class I on Normal Target Cells in Mice

Background

A major group of murine inhibitory receptors on Natural Killer (NK) cells belong to the Ly49 receptor family and recognize MHC class I molecules. Infected or transformed target cells frequently downmodulate MHC class I molecules and can thus avoid CD8⁺ T cell attack, but may at the same time develop NK cell sensitivity, due to failure to express inhibitory ligands for Ly49 receptors. The extent of MHC class I downregulation needed on normal cells to trigger NK cell effector functions is not known.

Methodology/Principal Findings

In this study, we show that cells expressing MHC class I to levels well below half of the host level are tolerated in an in vivo assay in mice. Hemizygous expression (expression from only one allele) of MHC class I was sufficient to induce Ly49 receptor downmodulation on NK cells to a similar degree as homozygous expression, despite a strongly reduced cell surface level of MHC class I. Co-expression of weaker MHC class I ligands in the host did not have any further effect on the degree of Ly49 downmodulation. Furthermore, a single MHC class I allele could downmodulate up to three Ly49 receptors on individual NK cells. Only when NK cells simultaneously expressed several Ly49 receptors and hemizygous MHC class I levels, a putative threshold for Ly49 downmodulation was reached.

Conclusion

Collectively, our findings suggest that in interactions between NK cells and normal untransformed cells, MHC class I molecules are in most cases expressed in excess compared to what is functionally needed to ensure self tolerance and to induce maximal Ly49 downmodulation. We speculate that the reason for this is to maintain a safety margin for otherwise normal, autologous cells over a range of MHC class I expression levels, in order to ensure robustness in NK cell tolerance.