A ligand for the murine NK activation receptor Ly-49D: activation of tolerized NK cells from beta 2-microglobulin-deficient mice

Mouse NK cells express inhibitory NK receptors that recognize target cell MHC class I molecules and activation receptors that are less well defined. The Ly-49D activation receptor on C57BL/6 NK cells recognizes Chinese hamster ovary cells and triggers natural killing. In this study, we demonstrate that a Chinese hamster classical MHC class I molecule is the ligand for Ly-49D in a reporter gene assay system as well as in NK cell killing assays. Ly-49D recognizes the Chinese hamster class I molecule better when it is expressed with Chinese hamster beta(2)-microglobulin (beta(2)m) than murine beta(2)m. However, it is still controversial that Ly-49D recognizes H-2D(d), as we were unable to demonstrate the specificity previously reported. Using this one ligand-one receptor recognition system, function of an NK activation receptor was, for the first time, investigated in NK cells that are tolerized in beta(2)m-deficient mice. Surprisingly, Ly-49D-killing activity against ligand-expressing targets was observed with beta(2)m-deficient mouse NK cells, albeit reduced, even though "tolerized" function of Ly-49D was expected. These results indicate that Ly-49D specifically recognizes the Chinese hamster MHC class I molecule associated with Chinese hamster beta(2)m, and indicate that the Ly-49D NK cell activation receptor is not tolerized in beta(2)m deficiency.     

HLA-B27 in inbred and non-inbred transgenic mice. Cell surface expression and recognition as an alloantigen in the absence of human beta 2-microglobulin

A gene encoding the H chain of the human class I MHC Ag HLA-B27 was introduced into the germ lines of inbred C57BL/6 (B6) and non-inbred (B6 X SJL/J) F2 mice. By immunofluorescence and flow cytometry, the HLA-B27 gene product was expressed on lymphoid cells at levels comparable to the endogenous H-2b and H-2s class I MHC molecules. In both primary and secondary MLC between responder spleen cells from non-transgenic (B6 X SJL/J) F1 mice and transgenic stimulator cells, CTL were generated that specifically lysed mouse L cell (H-2k) or human B cell targets expressing HLA-B27, and this lysis thus appeared largely unrestricted by H-2. These results indicate that transgenic mice express a functional HLA-B27 gene product on cell surfaces in the absence of the human beta 2-microglobulin gene. These transgenic mice promise to be a valuable resource in the investigation of the unique role of HLA-B27 in inflammatory human disease.     

Functional and phenotypic evidence for presentation of E alpha 52-68 structurally related self-peptide(s) in I-E alpha-deficient mice

The Y-Ae mAb and the 1H3.1 TCR-alpha beta (V alpha 1/V beta 6) are two immune receptors specific for I-Ab MHC class II molecules complexed to the 52-68 fragment of the alpha-chain of I-E class II molecules (the E alpha 52-68 peptide). A profound intrathymic negative selection occurs in 1H3.1 TCR transgenic mice in the presence of an I-E alpha transgene. The administration of mAbs to 1H3.1/I-E alpha double-transgenic newborn mice reveals that Y-Ae, but not the isotype-matched anti-I-E Y17 mAb, rescues a significant number of mature (V beta 6highCD4+CD8-) thymocytes and allows the detection of E alpha 52-68-reactive T cells in the periphery. These observations indicate that deletion of autoreactive T cells can be specifically inhibited in vivo by an mAb specific for the deleting self-peptide:self-MHC class II complex. Similar inhibition experiments indicate that C57BL/6 (I-Ab+/I-E alpha-) mice constitutively express an E alpha-independent, Y-Ae-recognizable epitope(s). This finding is confirmed by the phenotypic analysis of mature (MHC class II high) C57BL/6 bone marrow-derived dendritic cells. Collectively, these observations further illustrate the peptide specificity of negative selection and demonstrate that MHC class II-positive cells from unmanipulated C57BL/6 mice that lack a functional I-E alpha gene can assemble one or more self-peptide:I-Ab complexes recognizable by the E alpha 52-68:I-Ab complex-specific Y-Ae mAb.     

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.     

Cell-surface expression and alloantigenic function of a human nonclassical class I molecule (HLA-E) in transgenic mice

We have introduced the gene (E*01033) encoding the heavy chain of the human nonclassical MHC class I Ag, HLA-E, into the mouse genome. Two founder mice carry a 21-kb fragment, the others bear an 8-kb fragment. Each of the founder mice was mated to mice of an already established C57BL/10 transgenic line expressing human beta2-microglobulin (beta2m). Cell surface HLA-E was detected on lymph node cells by flow cytometry only in the presence of endogenous human beta2m. However, HLA-E-reactive mouse CTL (H-2-unrestricted) lysed efficiently the target cells originating from HLA-E transgenic mice without human beta2m, showing that the HLA-E protein can be transported to the cell surface in the absence of human beta2m, presumably by association with murine beta2m. Rejection of skin grafts from HLA-E transgenic mice demonstrates that HLA-E behaves as a transplantation Ag in mice. HLA-E transgenic spleen cells are effective in stimulating an allogeneic CTL response in normal and human classical class I (HLA-B27) transgenic mice. Furthermore, results from split-well analysis indicate that the majority of the primary in vivo-induced CTL recognizes HLA-E as an intact molecule (H-2-unrestricted recognition) and not as an HLA-E-derived peptide presented by a mouse MHC molecule, although a small fraction (ranging from 4 to 21%) of the primary in vivo-induced CTL is able to recognize HLA-E in an H-2-restricted manner. Based on these observations, we conclude that HLA-E exhibits alloantigenic properties that are indistinguishable from classical HLA class I molecules when expressed in transgenic mice.     

Transgene number-dependent, gene expression rate-independent rejection of Dd -, Kd-, or Dd Kd -transgened mouse skin or tumor cells from C57BL/6 Db Kb mice

Recipient cells migrating into the transplantation site of an allograft recognize histocompatibility antigens on the grafts and are cytotoxic against the grafts. Although the alloreactive immune response is predominantly directed at the major histocompatibility complex (major histocompatibility complex [MHC]; H-2 in mice) class I molecules, the basic mechanisms of allograft rejection (e.g., ligand-receptor interaction) remain unclear, because of the polymorphism and complexity of the MHC. To examine the role of MHC class I molecules in allograft rejection, D(d) , K(d) or D(d) K(d) -transgenic skin or tumor cells we established on a C57BL/6 (D(b) K(b) ) background and transplanted into C57BL/6 mice. Skin grafts from allogeneic (i.e., BALB/c, B10.D2, and BDF1) strains of mice were rejected from C57BL/6 mice on days 12-14 after grafting, whereas isografts were tolerated by these mice. Unexpectedly, skin grafts from D(d) -, K(d) -, and D(d) K(d) -transgenic C57BL/6 mice were rejected on days 12-14 in a transgene expression rate-independent manner from 9/19 (47%), 20/39 (51%), and 12/17 (71%) of C57BL/6 mice, respectively. Similarly, intradermally transplanted allogeneic (i.e., Meth A), but not syngeneic (i.e., EL-4), tumor cells were rejected from C57BL/6 mice; the growth of D(d) - or K(d) -transfected EL-4 cells was delayed by 10-13 days; and 4/10 (40%) of D(d) K(d) -transfected tumor cells were rejected from C57BL/6 mice. These results indicate that D(d) and K(d) genes are equivalent as allogeneic MHC class I genes and that C57BL/6 (D(b) K(b) ) mice reject D(d) -, K(d) -, or D(d) K(d) -transgened skin or tumor cells in a transgene number-dependent, gene expression rate-independent manner.     

Genetic differences in BCG-induced resistance to Schistosoma mansoni are not controlled by genes within the major histocompatibility complex of the mouse.

Induction of nonspecific resistance to Schistosoma mansoni infection after the i.v. injection of viable BCG was investigated in outbred mice and a panel of inbred and H-2 congenic strains. Significant protection was induced in CF1, A/J, C57BL/6, C57BL/10, DBA/2, C57BR, and SJL mice. BALB/c mice were not protected whereas CBA and C3H mice expressed intermediate degrees of protection. Expression of the protective phenomenon is not controlled by genes within the MHC as shown by the marked differences in response between BALB/c and DBA/2 (H-2d) as well as between C57BR and C3H (H-2k) mice. H-2 congenic strains with C57BL/10 background (B10.A and B10.D2) were high responders. BALB.B10 mice carrying the high responder (B10) MHC on the nonresponder (BALB/c) background were not protected. The degree of splenic hypertrophy did not correlate with the expression of nonspecific resistance. These results demonstrate that, in addition to controlling specific immune responses, genetic differences influence the nonspecific protective phenomena related to BCG administration as well.     

Social experience occurring during adult life: its effects on socio-sexual olfactory preferences in inbred mice,Mus musculus

Adult female mice of the inbred strain SEC1Re/J (SEC), either individually cross-fostered by the C57BL6/J (C57) strain or non-fostered, preferred, when naive, the scent of C57 males to that of SEC males. Following an exposure to males of either strain, which allowed acoustic and olfactory interactions to take place between the experimental females and the stimulus-males, those females that had experienced C57 male cues strengthened their preference for C57 male scent. By contrast, subjects exposed to SEC males preferred SEC male scent, so reversing their previous choice. Thus, the initial tendency to choose the odour of C57 males appeared to be modified by the effects of appropriate social experience during adulthood.     

Ly49-dependent NK cell licensing and effector inhibition involve the same interaction site on MHC ligands

NK cells become functionally competent to be triggered by their activation receptors through the interaction of NK cell inhibitory receptors with their cognate self-MHC ligands, an MHC-dependent educational process termed "licensing." For example, Ly49A(+) NK cells become licensed by the interaction of the Ly49A inhibitory receptor with its MHC class I ligand, H2D(d), whereas Ly49C(+) NK cells are licensed by H2K(b). Structural studies indicate that the Ly49A inhibitory receptor may interact with two sites, termed site 1 and site 2, on its H2D(d) ligand. Site 2 encompasses the α1/α2/α3 domains of the H2D(d) H chain and β(2)-microglobulin (β2m) and is the functional binding site for Ly49A in effector inhibition. Ly49C functionally interacts with a similar site in H2K(b). However, it is currently unknown whether this same site is involved in Ly49A- or Ly49C-dependent licensing. In this study, we produced transgenic C57BL/6 mice expressing wild-type or site 2 mutant H2D(d) molecules and studied whether Ly49A(+) NK cells are licensed. We also investigated Ly49A- and Ly49C-dependent NK licensing in murine β2m-deficient mice that are transgenic for human β2m, which has species-specific amino acid substitutions in β2m. Our data from these transgenic mice indicate that site 2 on self-MHC is critical for Ly49A- and Ly49C-dependent NK cell licensing. Thus, NK cell licensing through Ly49 involves specific interactions with its MHC ligand that are similar to those involved in effector inhibition.     

MHC odours are not required or sufficient for recognition of individual scent owners

To provide information about specific depositors, scent marks need to encode a stable signal of individual ownership. The highly polymorphic major histocompatibility complex (MHC) influences scents and contributes to the recognition of close kin and avoidance of inbreeding when MHC haplotypes are shared. MHC diversity between individuals has also been proposed as a primary source of scents used in individual recognition. We tested this in the context of scent owner recognition among male mice, which scent mark their territories and countermark scents from other males. We examined responses towards urine scent according to the scent owner's genetic difference to the territory owner (MHC, genetic background, both and neither) or genetic match to a familiar neighbour. While urine of a different genetic background from the subject always stimulated greater scent marking than own, regardless of familiarity, MHC-associated odours were neither necessary nor sufficient for scent owner recognition and failed to stimulate countermarking. Urine of a different MHC type to the subject stimulated increased investigation only when this matched both the MHC and genetic background of a familiar neighbour. We propose an associative model of scent owner recognition in which volatile scent profiles, contributed by both fixed genetic and varying non-genetic factors, are learnt in association with a stable involatile ownership signal provided by other highly polymorphic urine components.     

The MHC locus controls size variations in the CD4 compartment of the mouse thymus

The genetic factors that determine the size of lymphocyte populations are largely unknown. We studied the genetic control of variations in the size of the CD4 thymic compartment in unmanipulated mouse strains. A time-course experiment showed that the C57BL/6 mouse strain has a consistently reduced proportion of thymic CD4 cells compared to the BALB/c strain. This difference denotes a decrease in the efficiency of the transition from CD4(+)CD8(+) to CD4(+) thymocytes in the C57BL/6 mouse. Genome-wide genetic mapping identified a major quantitative trait locus in the MHC region that controlled the variations in the proportion of CD4 thymocytes in an F2 intercrossed progeny from C57BL/6 and BALB/c mice (LOD score=21.7). The linkage was maximal at the MHC class II molecule Ea locus, which explained 59% of the observed phenotypic variance. As the C57BL/6 mouse does not express Ea, we hypothesize that the decreased size of the CD4 compartment in the C57BL/6 thymus is due to a reduction in the number of functional MHC class II genes. This study suggests that, at the level of the thymus, the MHC molecules in addition to conferring functional restriction and self-tolerance on the T-cell repertoire, also play a role in determining the homeostasis of the thymic compartments.     

Functional expression of murine V2R pheromone receptors involves selective association with the M10 and M1 families of MHC class Ib molecules

The vomeronasal organ (VNO) of the mouse has two neuronal compartments expressing distinct families of pheromone receptors, the V1Rs and the V2Rs. We report here that two families of major histocompatibility complex (MHC) class Ib molecules, the M10 and the M1 families, show restricted expression in V2R-expressing neurons. Our data suggest that neurons expressing a given V2R specifically co-express one or a few members of the M10 family. Biochemical and immunocytochemical analysis demonstrates that in VNO sensory dendrites M10s belong to large multi-molecular complexes that include pheromone receptors and beta2-microglobulin (beta2m). In cultured cells, M10s appear to function as escort molecules in transport of V2Rs to the cell surface. Accordingly, beta2m-deficient mice exhibit mislocalization of V2Rs in the VNO and a specific defect in male-male aggressive behavior. The functional characterization of M10 highlights an unexpected role for MHC molecules in pheromone detection by mammalian VNO neurons.     

Single amino acid replacements in an antigenic peptide are sufficient to alter the TCR V beta repertoire of the responding CD8+ cytotoxic lymphocyte population.

Cytotoxic CD8+ T lymphocytes are activated upon the engagement of their Ag-specific receptors by MHC class I molecules loaded with peptides 8-11 amino acids long. T cell responses triggered by certain antigenic peptides are restricted to a limited number of TCR V beta elements. The precise role of the peptide in causing this restricted TCR V beta expansion in vivo remains unclear. To address this issue, we immunized C57BL/6 mice with the immunodominant peptide of the vesicular stomatitis virus (VSV) and several peptide variants carrying single substitutions at TCR-contact residues. We observed the expansion of a limited set of TCR V beta elements responding to each peptide variant. To focus our analysis solely on the TCR beta-chain, we created a transgenic mouse expressing exclusively the TCR alpha-chain from a VSV peptide-specific CD8+ T cell clone. These mice showed an even more restricted TCR V beta usage consequent to peptide immunization. However, in both C57BL/6 and TCR alpha transgenic mice, single amino acid replacements in TCR-contact residues of the VSV peptide could alter the TCR V beta usage of the responding CD8+ T lymphocytes. These results provide in vivo evidence for an interaction between the antigenic peptide and the germline-encoded complementarity-determining region-beta loops that can influence the selection of the responding TCR repertoire. Furthermore, only replacements at residues near the C terminus of the peptide were able to alter the TCR V beta usage, which is consistent with the notion that the TCR beta-chain interacts in vivo preferentially with this region of the MHC/peptide complex.     

Activation of murine T cells by staphylococcal enterotoxin E: requirement of MHC class II molecules expressed on accessory cells and identification of V beta sequence of T cell receptors in T cells reactive to the toxin

We investigated a mechanism leading to activation of murine T cells by staphylococcal enterotoxin E (SEE). L cells transfected with I-Ab genes but not control L cells supported IL-2 production by SEE-induced C57BL/6 T lymphoblasts upon restimulation with SEE. mAb to I-Ab markedly inhibited the above response. Flow cytometric analyses showed that SEE-induced C57BL/6 T lymphoblasts are composed of both CD4+ T cells and CD8+ T cells, and that larger parts of them bore V beta 11 (40-75%). mAb to V beta 11 markedly inhibited the SEE-induced proliferative response and IL-2 production by T cells. Analysis of SEE-induced IL-2 production in spleen cells from various mouse strains showed that C57BL/6 and B10.A(4R) mice (I-E, not expressed; V beta 11+ T cells, normally generated) are highly responsive to SEE. In contrast, BALB/c, C3H/HeN, (C57BL/6 x BALB/c or C3H/HeN) F1 mice (I-E, normally expressed and V beta 11+ T cells, deleted), and SJL and C57L mice (V beta 11 genes, deleted) are weakly responsive to SEE. The results indicate that SEE activates mainly T cells bearing V beta 11 in physical association with MHC class II molecules expressed on AC. In addition, the results indicate that SEE activates both CD4+ T cells and CD8+ T cells.     

Hepatocytes express abundant surface class I MHC and efficiently use transporter associated with antigen processing, tapasin, and low molecular weight polypeptide proteasome subunit components of antigen processing and presentation pathway

Hepatic expression levels of class I MHC Ags are generally regarded as very low. Because the status of these Ags and their ability to present peptides are important for the understanding of pathogen clearance and tolerogenic properties of the liver, we set out to identify the factors contributing to the reported phenotype. Unexpectedly, we found that the surface densities of K(b) and D(b) on C57BL/6 mouse hepatocytes are nearly as high as on splenocytes, as are the lysate concentrations of mRNA encoding H chain and beta(2)-microglobulin (beta(2)m). In contrast, the components of the peptide-loading pathway are reduced in hepatocytes. Despite the difference in the stoichiometric ratios of H chain/beta(2)m/peptide-loading machineries, both cell types express predominantly thermostable class I and are critically dependent on TAP and tapasin for display of surface Ags. Minor differences in the expression patterns in tapasin(-/-) background suggest cell specificity in class I assembly. Under immunostimulatory conditions, such as exposure to IFN-gamma or Listeria monocytogenes, hepatocytes respond with a vigorous mRNA synthesis of the components of the Ag presentation pathway (up to 10-fold enhancement) but up-regulate H chain and beta(2)m to a lesser degree (<2-fold). This type of response should promote rapid influx of newly generated peptides into the endoplasmic reticulum and preferential presentation of foreign/induced Ag by hepatic class I.     

Reduced scent marking and ultrasonic vocalizations in the BTBR T+tf/J mouse model of autism

Qualitative impairments in communication, such as delayed language and poor interactive communication skills, are fundamental to the diagnosis of autism. Investigations into social communication in adult BTBR T+tf/J (BTBR) mice are needed to determine whether this inbred strain incorporates phenotypes relevant to the second diagnostic symptom of autism, communication deficits, along with its strong behavioral phenotypes relevant to the first and third diagnostic symptoms, impairments in social interactions and high levels of repetitive behavior. The aim of the present study was to simultaneously measure female urine‐elicited scent marking and ultrasonic vocalizations in adult male BTBR mice, in comparison with a standard control strain with high sociability, C57BL/6J (B6), for the assessment of a potential communication deficit in BTBR. Adult male BTBR mice displayed lower scent marking and minimal ultrasonic vocalization responses to female urine obtained from both B6 and BTBR females. Lower scent marking and ultrasonic vocalizations in a social setting by BTBR, as compared with B6, are consistent with the well‐replicated social deficits in this inbred mouse strain. Our findings support the interpretation that BTBR incorporate communication deficits, and suggest that scent marking and ultrasonic vocalizations offer promising measures of interest in social cues that may be widely applicable to investigations of mouse models of autism.     

Critical residues at the Ly49 natural killer receptor's homodimer interface determine functional recognition of m157, a mouse cytomegalovirus MHC class I-like protein

NK cell function is regulated by Ly49 receptors in mice and killer cell Ig-like receptors in humans. Although inhibitory Ly49 and killer cell Ig-like receptors predominantly ligate classical MHC class I molecules, recent studies suggest that their activating counterparts recognize infection. The quintessential example is resistance to the mouse CMV in C57BL/6 mice, which depends on the functional recognition of m157, a mouse CMV-encoded MHC class I-like molecule, by Ly49H, an activating NK cell receptor. We have taken advantage of the natural variation in closely related members of the Ly49C-like receptors and the availability of Ly49 crystal structures to understand the molecular determinants of the Ly49H-m157 interaction and to identify amino acid residues discriminating between m157 binding and nonbinding receptors. Using a site-directed mutagenesis approach, we have targeted residues conserved in receptors binding to m157 (Ly49H and Ly49I(129)) but different from receptors lacking m157 recognition (Ly49C, Ly49I(B6), and Ly49U). Wild-type and mutant receptors were transfected into reporter cells, and physical binding as well as functional activation by m157 was studied. Our findings suggested that the Ly49 MHC class I contact "site 2," I226, may not be involved in m157 binding. In contrast, residue Y146 and G151, mapping at the receptor homodimer interface, are likely critical for functional recognition of the m157 glycoprotein. Our combined functional and three-dimensional modeling approach suggested that the architecture of the Ly49H dimer is crucial to accessing m157, but not MHC class I. These results link Ly49 homodimerization variability to the direct recognition of pathogen products.     

异体软骨移植及其兔疫原性研究近交系小鼠动物模型的建立

选用两组主要组织相容性复合体不相同的近交系小鼠Ｃ５７ＢＬ／６和ＤＢＡ／２做为本实验的动物模型,分别将异体软骨和同基因软骨移植于Ｃ５７ＢＬ／６小鼠股部肌肉中。通过检测抗体－补体介导的细胞杀伤反应和细胞介导的细胞杀伤反应,观察移植软骨的免疫原性。实验结果显示,异体软骨移植后产生强烈的特异性免疫排斥反应,而同基因软骨移植后不产生免疫排斥反应,与阴性对照组无显著性差异。故认为Ｃ５７ＢＬ／６和ＤＢＡ／２可以作为异体软骨移棺及其免疫原性研究的动物模型。