Biosynthesis of glycophospholipid bound and secreted murine class I Qa-2 polypeptides

Murine T cells synthesize and express a cell-surface glycophospholipid anchored 40 kDa and a secreted water-soluble 39 kDa Qa-2 polypeptide. We have examined the biosynthetic pathways which lead to the production of the membrane-bound and water-soluble isoforms of the Qa-2 molecule. Using the detergent TX-114, both detergent (membrane)-bound and soluble Qa-2 polypeptides can be identified in cell lysates and can be distinguished by charge and molecular weight. Two membrane-bound forms, a 40-kDa Endo H resistant cell-surface form and a 38 kDa-Endo H sensitive form can be identified, both of which can be biosynthetically labeled with 3H-ethanolamine and can be converted to water soluble forms by digestion with a phosphatidylinositol specific phospholipase C. In addition, several water soluble polypeptides at 39, 37, 35 kDa, and a minor species at 33 kDa were identified, none of which radiolabel with 3H-ethanolamine. While the 39-kDa polypeptide was Endo H resistant, the other isoforms were sensitive to Endo H digestion. Pulse chase experiments and molecular weights of the deglycosylated core polypeptides suggest a precursor to product relationship between the intracellular water-soluble species and the mature 39-kDa secreted Qa-2 molecule. This relationship is supported by the observation that murine L cells transfected with the Qa-2 encoding class I gene Q7 fail to express membrane-bound Qa-2 molecules yet synthesize both intracellular water-soluble and secreted Qa-2 molecules. These findings argue for a pathway in which secreted soluble Qa-2 molecules are derived from intracellular precursors.     

Expression of heterophile, Paul-Bunnell and Hanganutziu-Deicher antigens on human melanoma cell lines

Expression of heterophile antigens was studied on 6 human melanoma cell lines. Paul-Bunnell and Hanganutziu-Deicher (H-D) antigens, but not Forssman antigen, were demonstrated on these cell lines. H-D antigen was also demonstrated on melanoma cells isolated from primary and metastatic lesions. Evidence was also presented that HLA class I but not class II (DR and DQ) molecules are expressed on these cell lines. H-D antibodies of IgG class were detected in 10 of 23 (42%) melanoma patients suggesting the possibility that H-D antigen might have been expressed in an immunogenic form in the patients.     

Loss of expression of antigen-presenting molecules in human pancreatic cancer and pancreatic cancer cell lines

Tumours evade immune recognition and destruction through loss or down-regulation of expression of antigen processing and antigen-presenting molecules such as the human leucocyte antigen (HLA class I) and transporter for antigen presentation (TAP). This study examined the expression of HLA class I, class II and TAP in human pancreatic carcinoma tissue and 19 immortalized pancreatic cancer lines using a panel of antibodies directed against allele-specific as well as monomorphic determinants of these molecules. In tissue samples, reduction or loss of HLA class I and TAP was observed in 76% of cases, loss or down-regulation of TAP expression in 53%. In pancreatic cell lines down-regulation or loss of class I and TAP expression was also observed frequently. However, reductions in class I and TAP expression were reversible upon exposure to interferon-gamma in vitro, suggesting a regulatory rather than structural defect in these genes. De novo class II expression was observed in 26% of tumours and 42% of cell lines and may reflect the differentiation status of the cells. The high rate of class I and TAP loss has implications for immunotherapy strategies for pancreatic cancer, as such changes could facilitate a selective growth advantage for malignant cells. However, the reinduction of expression of these molecules with cytokines such as interferon-gamma may ultimately allow their cytotoxic T cell-mediated destruction.     

Soluble HLA class I antigen secretion by normal lymphocytes: relationship with cell activation and effect of interferon-gamma

HLA class I antigens are thought to be integral membrane proteins. However, soluble forms of these molecules have been detected. Our laboratory has recently shown that the predominant form of these soluble proteins present in human serum, spleen tissue and culture supernatant of activated lymphocytes exhibits molecular weight and structure similar to classical HLA class I antigens, but lacks HLA A or B polymorphic determinants. In the present study, the secretion of such soluble proteins by lymphocytes has been further explored. Phytohaemagglutinin-stimulated normal lymphocytes secrete considerable quantities of soluble HLA (sHLA) class I proteins. This secretion seems to be a general property of lymphocytes, since activation of T as well as B cells by appropriate mitogens equally induce sHLA I secretion. Lymphocytes require RNA and protein synthesis, but not DNA synthesis, for the secretion to occur. Kinetic studies reveal that maximal sHLA I secretion precedes the peak of DNA synthesis by 24 h. In vitro stimulation with antigens or alloantigens also provokes sHLA I secretion. Moreover, this phenomenon has also been detected for in vivo-activated lymphocytes, as enhanced spontaneous sHLA I secretion was observed in cultures of low-density blastic B and T cells, and of blood lymphocytes obtained from normal subjects who had received a booster immunization 5 days earlier. Interferon-gamma (IFN-gamma) increases the expression of membrane-bound class I antigens but does not induce any sHLA I secretion, suggesting that both molecules are under different regulatory mechanisms. Our results indicate that human lymphocytes, upon stimulation, actively secrete considerable amounts of a soluble form of these biologically relevant proteins.     

Interferon-gamma drives the metalloproteinase-dependent cleavage of HLA class I soluble forms from primary human bronchial epithelial cells

Activation of bronchial epithelial cells (BEC) and disruption of an intact epithelial barrier in a lung transplant recipient can lead to acute or chronic rejection, events that are associated with release of soluble human leukocyte antigen (sHLA) class I. Although we know that HLA is released from mitogen-activated lymphocytes in a metalloproteinase (MPase)-dependent fashion, the mechanism of release from nonlymphoid tissue is not well understood. To this end, we stimulated primary BEC with increasing amounts of the T-helper cell-1 cytokines, interferon gamma (IFNgamma), and/or tumor necrosis factor alpha (TNFalpha) and measured the quantity and forms of HLA class I release. We found that IFNgamma, but not TNFalpha, was able to stimulate a time- and concentration-dependent release of HLA/beta(2)m and beta(2)m-free heavy chain (HC) from the BEC. A portion (50%) of the HLA/beta(2)m release and >90% of the beta(2)m-free HC release was mediated by a MPase. Western blot analysis supported the conclusion that a MPase-sensitive pathway produced 36 and 37 kDa cleaved forms, whereas the secreted 39 kDa form of beta(2)m-associated soluble HLA class I (sHLA/beta(2)m) was MPase-resistant. This adds to the growing understanding of the extracellular processing pathways of major histocompatibility complex class I that may be critical for both chronic rejection as well as immune regulation.     

Circulating histocompatibility antigen (HLA) gene products may help differentiate benign from malignant indeterminate pulmonary lesions

Background

This study explores the potential diagnostic utility of soluble Human Leukocyte Antigen (sHLA) molecules differentially released by lung adenocarcinoma and benign lung lesions.

Biochemical analysis of plasma-soluble invariant chains and their complex formation with soluble HLA-DR

The invariant chain (CD74) is preferentially localized in the cytoplasm and regulates the loading of exogenous derived peptides into HLA class II heterodimers. In addition, a small proportion of CD74:class II complexes is also expressed on the cell surface. We identified and quantified soluble CD74 (sCD74) molecules in the plasma and sCD74:sHLA-DR complexes by ELISA. EDTA plasma samples from 86 healthy probands were analyzed. sCD74 could be detected in all samples with a mean concentration of 1.14 relative units±1.04 SD (range 0.17-4.31). Approximately 10% of the samples had increased amounts of sCD74 (3.0 relative units). Complexes of sCD74 and sHLA-DR were detected in all samples and their quantities were positively correlated (r=0.83, p0.001) with the sCD74 concentrations. SDS-PAGE analysis of plasma samples with high sCD74 concentrations (3.0 relative units) revealed four isoforms of sCD74 with molecular weights of 45, 43, 35, 31 kDa corresponding to known sizes of intracellular CD74. However, only molecular weights of the 45 and 43 kDa isoforms of sCD74 are found complexed with sHLA-DR. Our data demonstrate, that CD74 molecules are present in their soluble form in the plasma of healthy probands and form complexes with soluble HLA-DR molecules.     

Structure and function of the CD94 C-type lectin receptor complex involved in recognition of HLA class I molecules

Summary: A multigene family of immunoglobulin superfamily (Ig-SF) killer cell inhibitory receptors (KIRs) specifically recognize HLA class I molecules, while the interaction with H-2 products is mediated by members of the murine Ly49 C-type lectin family. A common structural feature of these receptors with inhibitory function is the presence of cytoplasmic immunoreceptor tyrosine-based inhibitory motifs (ITIMs) that couple them to SHP phosphatases. Strong support for the involvement of the CD94 C-type lectin receptor complex in NK cell-mediated recognition of Bw6+ HLA-B, HLA A and HLA-C alleles has been obtained. The cloned CD94 molecule covalently assembles with at least two different glyco-proteins (43 kDa and 39 kDa) to form functional receptors. NK cells inhibited upon HLA recognition express the CD94/p43 dimer, whose specificity for HLA molecules partially overlaps the Ig-SF receptor system. By contrast. NK clones bearing the homologous CD94/p39 receptor are triggered upon its ligation by CD94-specific mAbs. Remarkably, a set of Ig-SF receptors (p50) homologous to p58 KIRs also display an activating function. CD94-associated molecules belong to the NKG2 family of C-type lectins; the NKG2-A gene encodes for the p43 subunit. which contains cytoplasmic ITIMS. Expression of the different CD94 heterodimeric receptors will enable precise analysis of their putative interaction with HLA class I molecules.     

Mild acid treatment induces cross-reactivity of 4H84 monoclonal antibody specific to nonclassical HLA-G antigen with classical HLA class I molecules

Mild acid treatment by releasing beta(2)m and antigenic peptides leaves human leukocyte antigen (HLA) class I free heavy chains attached to the cell surface. Acid treatment thus allows detection of the cell surface class I antigens by monoclonal antibodies (mAbs) specific to HLA-free heavy chains. We found that acid treatment also enables detection of the cell surface non-classical HLA-G class I antigen with mAbs specific for HLA-G free heavy chains, including 4H84 mAb recognizing all isoforms. Furthermore, we found that 4H84 mAb, but not other mAbs specific to HLA-G free heavy chains, binds to the surface of 8 out of 16 acid-treated leukemia cell lines. Nevertheless, HLA-G antigen is not present in any of these leukemia cells. This was demonstrated by failure to detect any antigen with 4H84 mAb in immunoblotting as well as by inability to detect HLA-G mRNA by RT-PCR. The antigen recognized by 4H84 mAb in some acid treated leukemia cells was identified by immunoprecipitation as a 45 kDa protein. A number of observations indicate that 45 kDa proteins are none other than classical class I heavy chains. Acid treatment thus induces the ability of the 4H84 mAb to recognize some classical HLA class I molecules. Remarkably, 4H84 determinant on HLA-G is linear but corresponding determinant present on some partially folded classical HLA class I free heavy chains is conformational. In view of the unexpected cross-reactivity, detection of HLA-G with this mAb must be carefully evaluated to avoid false detection.     

Human leukocyte antigens (HLA) class I (Bg) on red cells studied with monoclonal antibodies

Monoclonal antibodies, capable of detecting monomorphic epitopes on HLA class I polypeptides and beta-microglobulin (Beta2-M), have been used by a variety of techniques to ascertain the type of structure detected on red blood cells (RBCs). Hemgglutination with class I monoclonal antibodies confirmed the reported relationship between Bg blood groups and HLA. It also established that the expression of HLA on RBCs which do not have nuclei is not normally strong, but may be enhanced in patients, notably those with systemic lupus erythematosus (SLE). Estimates of the number of class I molecules on mature RBCs by a radioligand-binding assay have confirmed that all HLA-B7 (Bga) individuals have higher numbers but that SLE patients usually have the most (124/RBC). Class I polypeptides were not elevated in the plasma of SLE patients and all RBCs lost molecules on aging in the circulation. These two facts suggest that HLA on RBCs is not acquired from plasma. when RBCs from SLE patients were immunoblotted with monoclonal antibodies, a complete 45 kDa intrinsic transmembrane heavy chain of HLA class I and a light chain of 11 kDa (Beta2-M) were detected. Chloroquine treatment and acid elution of RBCs did not remove HLA class I but only Beta2-M, As most antibodies recognize epitopes that depend on close association of class I with Beta2-M, the lost reactivity of treated RBCs may be understood.     

Expression of β2m-Free HLA Class I Heavy Chains in Neuroblastoma Cell Lines

Flow cytometry with the specific monoclonal antibody (MoAb) L31 was used to analyse the expression of HLA class I heavy chains not bound with β₂-microglobulin (β₂m) by neuroblastoma (NB) cell lines IMR-32 and LA-N-1. The cells, which express barely detectable amounts of β₂m-free (L31-positive molecules) and β₂m-complexed HLA class I antigens (W6.32- and BBM. I-reactive molecules), expressed MHC class I molecules not bound to light chains upon differentiation with either retinoic acid or serum starvation. The expression was not accompanied by an increase of surface heterodimers. Conversely, recombinant interferon-γ (rIFN-γ) treatment led IMR-32 and LA-N-1 cells to almost exclusively express β₂m-complexed HLA class I heavy chains. Surface β₂m-free MHC class I molecules displayed a molecular mass of ~45 kDa and did not bind exogenously added β₂m. No changes in the synthesis of either HLA class I and β₂m mRNAs or of L31 proteins were observed in differentiated NB cells, thus suggesting that the surface exposure of unusual HLA class I antigens is regulated post-translationally. These findings indicate that, in addition to activated lymphocytes, the surface expression of β₂m-free class I heavy chains is a feature of other cell types, such as NB cells.     

Phenotypic and functional characterization of a new human macrophage cell line K1m demonstrating immunophagocytic activity and signalling through HLA class II.

A human macrophage line, designated K1m, has been established from peripheral blood. K1m expresses a number of lineage-specific markers as well as a broad array of intercellular adhesion molecules. In particular, K1m expresses high levels of human leucocyte antigen (HLA) class I and class II. In response to ligation of HLA class II (HLA-DR), but not in response to ligation of HLA class 1, K1m forms tighter homotypic aggregates and develops a striking ''stellate'' culture phenotype. K1m also expresses Fc receptors for immunoglobulin G (IgG) (CD64, CD32, and CD16) and can be shown to phagocytose polystyrene latex beads, as well as neuroblastoma cells in the presence of tumour-specific monoclonal antibody (mAb). The K1m cell line should therefore prove useful for studying both signalling through macrophage HLA class II and immunophagocytosis.     

Natural killer susceptibility is independent of HLA class I antigen expression on cell lines obtained from human solid tumors.

The susceptibility to natural killer (NK) cell-mediated cytotoxicity of 20 cell lines obtained from human solid tumors and their class I histocompatibility antigen (HLA) levels were studied in an attempt to determine whether major histocompatibility complex (MHC) products expressed on cells derived from human solid tumors influence NK susceptibility. The effect of interferon-gamma (IFN-gamma) treatment on these elements was also analyzed. The MHC class I (HLA-ABC, HLA-A and HLA-B) antigen levels and degree of NK lysis were very heterogeneous and no correlation was found on comparison. After treatment with IFN-gamma a marked decrease in NK susceptibility was observed in all the cell lines, including the control line K-562. However, the level of HLA class I expression was not modified in any of the lines with the exception of the K-562, which increased. In some cell lines the expression of HLA class I-like antigens. CDla, b and c, was also measured before and after IFN treatment; however, no correlation was found between CD1 levels and NK susceptibility. Consequently, from our results it is possible to conclude that HLA class I antigens do not play a decisive role in NK susceptibility of cell lines derived from human solid tumors and to suggest that molecules which are not HLA class I antigens but IFN-gamma inducible may confer NK resistance to these lines.     

Soluble HLA-A,-B,-C and -G molecules induce apoptosis in T and NK CD8+ cells and inhibit cytotoxic T cell activity through CD8 ligation

There is convincing evidence that soluble HLA-A,-B,-C (sHLA-A,-B,-C) and soluble HLA-G (sHLA-G) antigens can induce apoptosis in CD8(+) activated T cells although there is scanty and conflicting information about the mechanism(s) by which sHLA-A,-B,-C antigens and sHLA-G antigens induce apoptosis. In this study we have compared the apoptosis-inducing ability of sHLA-A,-B,-C antigens with that of sHLA-G1 antigens in CD8(+) T lymphocytes and CD8(+) NK cells. Furthermore we have compared the inhibitory effect of sHLA-A,-B,-C antigens and of sHLA-G1 antigens on the activity of EBV-specific CD8(+) cytotoxic T lymphocytes (CTL). sHLA molecules were purified from serum and from the supernatant of HLA class I-negative cells transfected with one gene encoding either classical or non-classical HLA class I antigens. Both classical and non-classical sHLA class I molecules trigger apoptosis in CD8(+) T lymphocytes and in CD8(+) NK cells, which lack the T cell receptor, and their apoptotic potency is comparable. The binding of sHLA-A,-B,-C and sHLA-G1 molecules to CD8 leads to Fas ligand (FasL) up-regulation, soluble FasL (sFasL) secretion and CD8(+) cell apoptosis by Fas/sFasL interaction. Moreover, classical and non-classical sHLA class I molecules inhibit the cytotoxic activity of EBV-specific CD8(+) CTL. As the amount ofsHLA-G molecules detectable in normal serum is significantly lower than that of sHLA-A,-B,-C molecules, the immunomodulatory effects of sHLA class I molecules purified from serum are likely to be mainly attributable to classical HLA class I antigens. As far as the potential in vivo relevance of these findings is concerned, we suggest that classical sHLA class I molecules may play a major immunoregulatory role in clinical situations characterized by activation of the immune system and elevated sHLA-A,-B,-C serum levels. In contrast, non-classical HLA class I molecules may exert immunomodulatory effects in particular conditions characterized by elevated sHLA-G levels such as pregnancy and some neoplastic diseases.     

Beta2-micro-free HLA class I heavy chain levels in sera of healthy individuals. Lack of association with beta2-micro-associated HLA class I heavy chain levels and HLA phenotype

We have applied a double-determinant immune assay (DDIA) to measure soluble beta2-microglobulin (beta2-micro)-free HLA class I heavy chains in serum. The mean concentration of beta2-micro-free HLA class I heavy chains in serum from 120 healthy subjects was 0.21+/-0.24 microg/ml. The individual serum levels of beta2-micro-free HLA class I heavy chains had a wide distribution, did not seem to be related with HLA phenotype, were stable over time and did not change with age. The serum levels of soluble beta2-micro-free HLA class I heavy chains did not correlate with those of soluble beta2-micro-associated HLA class I heavy chains, suggesting that their release is independently regulated. Three forms of soluble beta2-micro-free HLA class I heavy chains, with apparent molecular masses of 44, 39 and 37-35 kD, respectively, circulate in human serum. These results provide a useful background to assess the serum level of soluble beta2-micro-free HLA class I heavy chains in pathological conditions and to evaluate their putative immunoregulatory function.     

C-terminal epitope tagging facilitates comparative ligand mapping from MHC class I positive cells

Purification of specific class I molecules prior to peptide ligand characterization is complicated by the presence of multiple class I proteins in most cell lines. Immortalized B, T, and tumor cell lines typically express endogenous HLA-A, -B, and -C; and most individuals from which the cell lines are derived are heterozygous at these loci. Antibodies specific for a particular HLA molecule may be used for purification, but allele-specific antibodies can be biased by ligands occupying the peptide-binding groove. Through the use of C-terminal tagging, we have developed a method of soluble HLA production such that downstream purification does not skew the peptide analysis of the examined molecule. Comparison of peptides eluted from HLA class I molecules with and without C-terminal tags demonstrates that addition of a tag does not abrogate the peptide binding specificity of the original molecule. Both pooled Edman sequencing and mass spectrometric sequencing identified no substantial differences in peptides bound by untailed, 6-HIS-tailed, and FLAG-tailed class I molecules, demonstrating that the peptide specificity of a given molecule is not distorted by either tag. This production methodology bypasses problems with isolation of specific molecules and permits ligand mapping and epitope discovery in a variety of pathogen-infected and tumor cell lines.     

Role of class I human leukocyte antigen molecules in early steps of echovirus infection of rhabdomyosarcoma cells

Several echoviruses use decay accelerating factor (DAF) as a cell surface receptor. However, most of them require additional cell surface coreceptors. We investigated the respective roles of DAF and class I human leukocyte antigen (HLA) molecules in the early steps of the echovirus 11 (EV11) lifecycle in rhabdomyosarcoma (RD) cells. EV11 infection was inhibited at an early stage by anti-β2-microglobulin (β2m) and anti-HLA monoclonal antibodies and by a soluble monochain HLA class I molecule. Expression of class I HLA molecules restored the early steps of the EV11 lifecycle, but its expression was not sufficient for EV11 replication and particle production. Expression of HLA class I molecules was associated with leukocyte cell line permissiveness to EV11 infection. In conclusion, HLA class I molecules are involved in the early steps of EV11 infection of RD cells and appear to participate in a complex interplay of surface molecules acting as coreceptors, including DAF.