Strategies for the measurements of expression levels and half-lives of HLA class I allotypes

HLA class I molecules are highly polymorphic cell surface proteins that trigger immune responses by CD8⁺ T cells and natural killer (NK) cells. Most humans express six different HLA class I proteins encoded by the HLA-A, HLA-B and HLA-C genes. HLA class I molecules bind to peptide antigens and present these antigens to T cell receptors (TCR) of CD8⁺ T cells. HLA class I expression levels also regulate NK cell activation. The presence of individual HLA class I genes is linked to many different disease, transplantation and therapy outcomes. An understanding of HLA class I expression and stability patterns is fundamentally important towards a better understanding of the associations of HLA class I genes with disease and treatment outcomes, and towards HLA class I targeting for vaccine development. Quantitative flow cytometry allows for assessments of variations in expression levels of HLA class I molecules in cells from a single blood donor over time, as well as averaged measurements across donors for the same allotype. Since all HLA class I molecules are structurally-related, cellular measurements of the HLA class I expression levels and stabilities of individual variants in human cells require careful choices of donors and antibodies, which are discussed here.     

Shaping the human NK cell repertoire: an epigenetic glance at KIR gene regulation

Human NK cells are equipped with arrays of inhibitory and stimulatory KIR receptors, many of them specific for HLA class I molecules on target cells. These NK receptors enable the recognition of virally infected as well as malignantly transformed target cells, which have downregulated the expression of single or multiple HLA class I products. KIR are expressed in clonally distributed ways leading to highly individualized but stable NK cell repertoires. Here, progress is reviewed toward understanding the molecular mechanisms that govern the unusual expression characteristics of KIR genes. Recent results suggest that DNA methylation plays a crucial role in shaping the KIR repertoire and underline the importance of epigenetic mechanisms as regulatory switches in the immune system.     

Natural killer cells lyse autologous herpes simplex virus infected targets using cytolytic mechanisms distributed clonotypically

Natural killer (NK) cells have the capability of lysing targets that have down-regulated the expression of HLA class I molecules. Herpes simplex virus (HSV) infection results in a profound reduction of HLA class I molecules on the surface of infected cells. For this reason, NK cell populations kill efficiently HSV-infected cells. The recent availability of a panel of monoclonal antibodies directed to NK receptors for HLA class I (CD158a, CD158b, anti-p70, anti-p140, and CD94) allowed an accurate dissection of the NK cell subpopulations. Using this approach, the relationship between the expression of NK cell receptors and the capability of lysing HSV-infected cell targets was analyzed at the clonal level. NK cell clones were derived from healthy donors, and cytolytic properties were assayed against HSV-infected autologous fibroblasts. NK cell clones, classified according to the expression of natural killer-cell receptors on their surface, displayed a great heterogeneity of cytolytic properties against HSV-infected cells. Nevertheless, a more accurate functional analysis demonstrated not only that HSV infection downregulated the expression of HLA-A and HLA-B and did not modify the expression of HLA-C, but also that NK cell clones expressing the "activating" form of the anti HLA-C NK cell receptor were more cytolytic than other clones. This finding suggests that two different and clonally distributed mechanisms of NK cell activation may be employed by NK cells to kill HSV-infected autologous target cells.     

Divergent effects of thyroid HLA-antigens on T and natural killer (NK) cell cytotoxicity.

We have used non-autoimmune non-neoplastic human thyroid cells to explore the role of surface class I and DR antigens on these cells' sensitivity towards T and Natural Killer (NK) cell cytotoxicity. Non-treated thyrocytes expressed class I but no DR antigens. Following incubation with gamma-interferon (gamma-IFN) class I antigens were markedly elevated and DR expression was induced. Whereas non-treated thyrocytes were minimally lysed by sensitized T cells, they served as appropriate targets for NK cells. Following incubation with gamma-IFN, the thyroid cells became highly sensitive to T cell lysis, with no significant reduction in their vulnerability to NK cell killing. The addition of monoclonal anti class I or DR antigens, or brief acid treatment which specifically eliminates class I molecules, inhibited T cell cytotoxicity but enhanced the sensitivity to lysis by NK cells. Thus, the presence of HLA antigens on the same thyroid cells have an opposite effect on two major cytotoxi mechanisms. Our findings are relevant within the context of recent suggestions of intervening with target HLA antigens for the management of autoimmune and malignant diseases.     

Killer immunoglobulin-like receptors

Killer Ig-like receptors (KIRs) are surface inhibitory receptors specific for allelic forms of human leukocyte antigen (HLA) class I molecules, which are expressed by natural killer (NK) cells and a subset of T lymphocytes. Upon engagement with HLA class I molecules, KIRs block NK cell activation and function. Cells lacking HLA class I molecules are promptly killed by NK cells because of the predominant effect of several activating NK receptors. The NK-mediated killing of these cells might represent an important defence mechanism, antagonizing spreading of pathogens and tumours. Evidence has been accumulated that KIR-encoding genes have evolved and diversified rapidly in primates and in humans. Similar to HLA loci, KIR sequences are highly polymorphic and, moreover, KIR haplotypes greatly vary in the number of the type of genes they contain. KIR gene expression is regulated by mechanisms of DNA methylation. As recently shown, the HLA class I regulated control of NK cell function can be exploited in an allogeneic bone marrow transplantation setting to eradicate acute myeloid leukaemias.     

Cellular ligands of activating NK receptors

Human natural killer (NK) cells are equipped with a series of surface receptors that recognise different cellular ligands on potential target cells. Some of these ligands [e.g. human leukocyte antigen (HLA) class I] prevent an NK-mediated attack by interacting with inhibitory NK receptors (e.g. killer Ig-like receptors). Other ligands interact with activating NK receptors that, once engaged, induce both cytotoxicity and lymphokine release. Tumour transformation (or viral infection) frequently results in downregulation of surface HLA class I molecules together with upregulation or de novo expression of ligands of triggering NK receptors. Thus, transformed cells can become highly susceptible to NK-mediated lysis. However, although NK cells use different means to identify and fight target cells, target cells have various strategies to hide themselves, and disarm or even confuse the immune system.     

Antiviral NK cell responses in HIV infection: I. NK cell receptor genes as determinants of HIV resistance and progression to AIDS

NK cells play an important role in controlling viral infections. They can kill virus-infected cells directly as well as indirectly via antibody-dependent, cell-mediated cytotoxicity. They need no prior sensitization and expansion for this killing. NK cells are also considered as important regulators of antiviral immune responses. They do so by secreting a multitude of soluble mediators and by directly interacting with other immune cells, e.g., dendritic cells. NK cells do not possess a single well-defined receptor to recognize antigens on target cells. Instead, they express an array of inhibitory and activating receptors and coreceptors, which bind to their cognate ligands expressed on the surface of target cells. These ligands include classical and nonclassical MHC class I antigens, MHC-like proteins, and a variety of other self- and virus-derived molecules. They may be expressed constitutively and/or de novo on the surface of virus-infected cells. NK cell receptors (NKRs) of the killer-cell Ig-like receptor (KIR) family, like their MHC class I ligands, are highly polymorphic. Several recent studies suggest that epistatic interactions between certain KIR and MHC class I genes may determine innate resistance of the host to viral infections, including HIV. In the first part of this review article, we provide an overview of the current state of knowledge of NK cell immunobiology and describe how NKR genes, alone and in combination with HLA genes, may determine genetic resistance/susceptibilty to HIV infection and the development of AIDS in humans.     

Specificity and function of immunoglobulin superfamily NK cell inhibitory and stimulatory receptors

Summary: Human NK cells express clonally distributed receptors specific for HLA-A, -B and -C molecules. These receptors belong to the immunoglobulin superfamily and can be functionally distinguished as inhibitory or stimulatory Inhibitory receptors block NK-cell-mediated cytotoxicity upon binding to HLA class I ligands. This function is mediated by phosphorylation of cytoplasmic tyrosines, which recruit the protein tyrosine phosphatase SHP-1. Stimulatory receptors also bind HLA class I, lack cytoplasmic tyrosine-based motifs, and trigger NK cytotoxicity and proliferation. Both types of receptor are characterized by a limited diversity allowing for recognition of distinct class I supertypic epitopes. This limited diversity is counterbalanced by the expression of different combinations ill inhibitory and stimulatory receptors with self and/or non-self HLA class I specificities on distinct NK cell clones. This peculiar strategy allows NK cells to detect loss of MHC class I molecules on autologous transformed and virally infected cells with maximal sensitivity.     

HCMV glycoprotein US6 mediated inhibition of TAP does not affect HLA-E dependent protection of K-562 cells from NK cell lysis

Human cytomegalovirus has evolved multiple strategies to interfere with immune recognition by the host. A variety of mechanisms affect antigen presentation by major histocompatibility complex class I molecules resulting in a reduced class I cell-surface expression. This downregulation is expected to trigger natural killer (NK) cytotoxicity, requiring counteraction by the virus to establish long-term infection. Here we describe that the human cytomegalovirus gpUS6 protein, which has been demonstrated to downregulate the expression of human leukocyte antigen (HLA) class I and the presentation of cytotoxic T lymphocyte epitopes by blocking transporter associated with antigen presentation (TAP function), does not affect the ability of HLA-E to inhibit NK cell mediated lysis of K-562 cells by interaction with CD94/NKG2A expressed on NK cells. Cell surface expression and function of HLA-E is not altered although gpUS6 inhibits TAP-dependent peptide transport by 95%. Moreover, HLA-E molecules presenting HLA class I signal sequence-derived peptides are functionally detectable on transfected TAP-deficient RMA-S cells.     

Structural insights into activation of antiviral NK cell responses

Natural killer (NK) cells are key components of innate immune responses, providing surveillance against cells undergoing tumorigenesis or infection, by viruses or internal pathogens. NK cells can directly eliminate compromised cells and regulate downstream responses of the innate and acquired immune systems through the release of immune modulators (cytokines, interferons). The importance of the role NK cells play in immune defense was demonstrated originally in herpes viral infections, usually mild or localized, which become severe and life threatening in NK-deficient patients 1 . NK cell effector functions are governed by balancing opposing signals from a diverse array of activating and inhibitory receptors. Many NK receptors occur in paired activating and inhibitory isoforms and recognize major histocompatibility complex (MHC) class I proteins with varying degrees of peptide specificity. Structural studies have made considerable inroads into understanding the molecular mechanisms employed to broadly recognize multiple MHC ligands or specific pathogen-associated antigens and the strategies employed by viruses to thwart these defenses. Although many details of NK development, signaling, and integration remain mysterious, it is clear that NK receptors are key components of a system exquisitely tuned to sense any dysregulation in MHC class I expression, or the expression of certain viral antigens, resulting in the elimination of affected cells.     

MHC antigens on human tumors.

MHC class I antigens on tumor cells are expected to play an important role because they regulate the sensitivity to antitumoral immunological mechanisms. Overall or selective qualitative or quantitative changes in MHC molecules may modify the recognition of tumor cells by components of the immune system. It seems clear that MHC antigen expression on tumor cells is important in triggering the immune response by autologous lymphocytes. A deficiency in or lack of MHC class I antigens may have profound effects on T and NK cell activity. In experimental models, variation in the expression of MHC class I antigens has been shown to exert a decisive influence on local tumor growth and metastasis. However, there is little information about the influence of selective loss of individual locus products on the behavior of human tumor cells. Total and selective HLA losses have been found in a large variety of tumors, and different mechanisms have been shown to be responsible for these changes. In some examples, HLA losses are associated with a poor degree of tissue differentiation and poor prognosis. In other tumors, however, no such association has been found. We do not know whether HLA class II expression in neoplastic cells plays an immunological role, although, with the exception of melanoma, HLA class II expression is more frequently observed in tumors with a more favorable prognosis. Finally, there is no doubt that we need to learn more about how to manipulate the expression of MHC class I and II antigens in human tumors, in order to stimulate immune response.(ABSTRACT TRUNCATED AT 250 WORDS)     

Major histocompatibility antigen expression in the liver in acquired immunodeficiency syndrome

Human leukocyte antigen (HLA) class I molecules are normally detected on most nucleated cells, but not on hepatocytes, while the expression of HLA class II antigens is mainly restricted to certain cell types of the immune system. This normal distribution pattern may be altered in human liver in a variety of disorders, particularly in infectious and immune diseases. In view of multiple infections and severe immune alterations in acquired immunodeficiency syndrome (AIDS), we studied the expression of HLA class I and class II antigens in liver obtained at autopsy from ten patients with AIDS and eight control patients, using a panel of monoclonal antibodies and the indirect immunofluorescence method. In spite of viral bacterial, and fungal infections, HLA class I antigens were detected on hepatocytes in only two patients with AIDS. Human leukocyte antigen class II antigens were found unexpectedly on bile duct epithelium in five of ten patients with AIDS and in one of eight control patients. The aberrant HLA class II antigen expression on bile ducts does not appear to be linked to bile duct damage or infection with cytomegalovirus or other agents, but may be related to immune alterations in patients with AIDS.     

Viral infection induces cytokine release by beta islet cells.

Viral infection has been suggested to play a triggering role in the pancreatic beta cell destruction which occurs in insulin-dependent diabetes (IDDM). However, the underlying mechanism of this phenomenon is unknown. In this study a human insulinoma cell line has been infected with measles, mumps and rubella viruses since a temporal association is reported between the clinical onset of IDDM and diseases caused by these viruses. The infection with measles and mumps viruses induced the release of interleukin-1 (IL-1) and interleukin-6 (IL-6) by the cell line as assessed by a bioassay and up-regulated the expression of human leucocyte antigen (HLA) class I and class II antigens as evaluated by cytofluorimetric analysis. Stimulation with rubella virus induced the release of IL-6 only and had no effect on HLA antigen expression. These data show for the first time that IL-1 and IL-6 secretion by an insulinoma cell line may occur after viral infection and suggest that cytokine release and increased expression of HLA molecules by beta cells may act to induce the immune response towards beta cells in IDDM.     

Increased serum concentration of soluble HLA-DR antigens in HIV infection and following transplantation

Abstract: HLA class I and class II antigens circulate in serum as soluble molecules. Increased concentrations of soluble HLA class I molecules have been demonstrated in viral diseases, in rejection episodes following organ transplantation and in graft versus host disease. To explore the possibility of a variation of the serum concentrations of soluble HLA class II molecules in the same pathologic conditions we developed a double determinant immune assay that detects whole soluble HLA-DR molecules (sHLA-DR). The mean level of sHLA-DR antigens in sera from 23 healthy individuals was 0.64±0.72 μg/ml. Elevated serum concentrations of sHLA-DR molecules were detected in sera from HIV infected patients in CDC2/3 and in CDC4C1 stages (2.0±1.7 g/ml and 4.6±1.7 g/ml, respectively), in sera from patients affected by acute rejection after liver transplantation (5.3±3.7 μg/ml) and in sera from patients affected by severe acute graft versus host disease following bone marrow transplantation (8.8±3.1 μg/ml). The increase of sHLA-DR molecules in these sera significantly correlated with the elevation of soluble HLA class I antigens (P=0.0004). The reported data suggest that both soluble HLA class I and class II molecules serum levels increase during viral infections and strong immune reactions and could suggest the involvement of these molecules in immunoregulation.     

Enhancement of class I HLA antigen expression by cytomegalovirus: role in amplification of virus infection

We have investigated the effect of cytomegalovirus (CMV) infection on the expression of class I HLA antigens on fibroblasts in vitro. Scanning and integrating microdensitometry was used to quantitate the level of cytoplasmic class I antigen expression, and an antibody binding assay was used to quantitate cell surface expression of class I HLA molecules. CMV infection resulted in a significant increase in the level of cytoplasmic and cell surface class I HLA antigen expression of fibroblast monolayers. The maximal effect was seen at 72 hours postinfection and was observed with both the laboratory strain of CMV, strain AD169, and with CMV purified directly from clinical specimens. Part of the increased HLA expression was mediated by interferon released from infected cells; however, an additional direct effect of the virus itself has not been ruled out. Interferon-induced increased expression of class I HLA antigens was accompanied by increased binding of CMV to the cells, consistent with our recent demonstration that class I HLA molecules can function as a cellular receptor for CMV.     

Structure/function of human killer cell immunoglobulin-like receptors: lessons from polymorphisms, evolution, crystal structures and mutations

Stimulation or tolerance of natural killer (NK) cells is achieved through a cross-talk of signals derived from cell surface activating and inhibitory receptors. Killer cell immunoglobulin-like receptors (KIR) are a family of highly polymorphic activating and inhibitory receptors that serve as key regulators of human NK cell function. Distinct structural domains in different KIR family members determine function by providing docking sites for ligands or signalling proteins. Here, we review a growing body of literature that has identified important structural elements on KIR that contribute to function through studies of engineered mutants, natural polymorphic sequence variants, crystal structure data and the conservation of protein sequences throughout primate evolution. Extensive natural polymorphism is associated with both human KIR and their ligands, MHC class I (HLA-A, -B and -C) molecules, and numerous studies have demonstrated associations between inheritance of certain combinations of KIR and HLA genes and susceptibility to several diseases, including viral infections, autoimmune disorders and cancers. In addition, certain KIR/HLA combinations can influence pregnancy and the outcome of haematopoietic stem cell transplantation. In view of the significant regulatory influences of KIR on immune function and human health, it is essential to fully understand the impacts of these polymorphic sequence variations on ligand recognition, expression and function of the receptor.