Distribution of the MHC antigens after liver transplantation: relationship with biochemical and histological parameters

The aim of this study was to analyse the change in tissue distribution of HLA class I and class II molecules in patients during the course of liver transplantation. Sixty-one liver biopsies were analysed in 24 patients with or without rejection (acute or chronic) episode. In 93% of cases with chronic rejection and in 48% of cases with acute rejection episodes, HLA class I antigens are expressed in the membrane and/or the cytoplasm of hepatocytes. The expression of these molecules was significantly correlated with the ASP (aspartate amino transferase), AP (alkaline phosphatase), the bile duct damage and the centrolobular lesions. In contrast, the expression of HLA class II molecules is not correlated with either the type of rejection, the biological or histological findings. A strong expression of HLA class I antigen on hepatocytes after day 60 may be one of the best signs of chronic rejection.     

HLA antigen and NK cell activating ligand expression in malignant cells: a story of loss or acquisition

Malignant transformation of cells is often associated with changes in classical and non-classical HLA class I antigen, HLA class II antigen as well as NK cell activating ligand (NKCAL) expression. These changes are believed to play a role in the clinical course of the disease since these molecules are critical to the interactions between tumor cells and components of both innate and adaptive immune system. For some time, it has been assumed that alterations in the expression profile of HLA antigens and NKCAL on malignant cells represented loss of classical HLA class I antigen and induction of HLA class II antigen, non-classical HLA class I antigen and/or NKCAL expression. In contrast to these assumptions, experimental evidence suggests that in some cases dysplastic and malignant cells can acquire classical HLA class I antigen expression and/or lose the ability to express HLA class II antigens. In light of the latter findings as well as of the revival of the cancer immune surveillance theory, a reevaluation of the interpretation of changes in HLA antigen and NKCAL expression in malignant lesions is warranted. In this article, we first briefly describe the conventional types of changes in HLA antigen and NKCAL expression that have been identified in malignant cells to date. Second, we discuss the evidence indicating that, in at least some cell types, classical HLA class I antigen expression can be acquired and/or the ability to express HLA class II antigens is lost. Third, we review the available evidence for the role of immune selective pressure in the generation of malignant lesions with changes in HLA antigen expression. This information contributes to our understanding of the role of the immune system in the control of tumor development and to the optimization of the design of immunotherapeutic strategies for the treatment of cancer.     

Modulation of HLA expression in human cytomegalovirus immune evasion

Human cytomegalovirus （hCMV） has evolved multiple mechanisms to escape the host immune recognition and innate or adaptive immune responses. Among them, hCMV has developed strategies to modulate the expression and/or function of human leukocyte antigens （HLAs）, including by encoding series of infection stage-dependent hCMV proteins to detain and destroy the expression of HLA molecules on the surface of infected cells. This disturbs the antigen presentation and processing, by encoding MHC class I homologues or selective up-regulation of particular HLA class I molecules binding to NK cell inhibitory receptors, and by encoding specific ligand antagonists to interfere with NK cell activating receptors. Here we discussed the molecular mechanisms utilized by the hCMV to alter the formation, transportation and expression of HLA antigens on the infected cell surface. The knowledge about hCMV modulating HLA expression could benefit us to further understand the pathogenesis of viral diseases and may eventually develop novel effective immunotherapies to counteract viral infections and viral associated diseases.     

Major histocompatibility complex status in breast carcinogenesis and relationship to apoptosis

Major histocompatibility complex (MHC) molecules are of central importance in regulating the immune response against tumors. In this study we used immunohistochemistry to study human leukocyte antigen (HLA) class I and II antigen expression in normal breast tissues and benign, preneoplastic, primary, and metastatic breast lesions using antibodies against beta-2-microglobulin (beta2-m), heavy-chain, and HLA-DR antigens. Whereas all normal tissues and benign lesions were positive for beta2-m and HLA-A, -B, and -C antigens, total loss of HLA class I antigens was found in 37% (11 of 30) of in situ carcinomas, in 43% (56 of 131) of the primary tumors, and in 70% (31 of 45) of the lymph node metastases. HLA-DR was also underexpressed in breast cancer cells; thus 20% (6 of 30) of in situ carcinomas, 15% of invasive carcinomas (20 of 131), and only 1 metastatic case were positive for this antigen. Both HLA class I and II antigen expression were more frequently down-regulated in metastatic lesions than in primary breast lesions (P <0.05), and a tendency toward a simultaneous defective expression of HLA class I and II antigens was observed in primary carcinomas (P = 0.07). However, no correlation was found between the expression of any of the aforementioned molecules and pathological parameters or survival. Interestingly, HLA class I expression was expressed more frequently in tissues with high apoptotic activity and was significantly associated with the expression of the proapoptotic bax gene (P = 0.02), and was inversely associated with expression of the antiapoptotic bcl-2 gene (P = 0.03). We conclude that alterations in HLA class I and II antigen expression are early events in breast carcinogenesis and play significant roles in metastatic progression. In addition, their expression is correlated with apoptosis-regulating proteins, which may influence the cytotoxicity of T cells against HLA class I-specific tumor antigens.     

Generic human leukocyte antigen class II (DRB1 and DQB1) alleles in patients with paracoccidioidomycosis.

Human leukocyte antigen (HLA) class II alleles are involved in antigen processing and in the presentation of antigens to T lymphocytes. Few studies have investigated HLA genes in paracoccidioidomycosis. In the present investigation, we analyzed the distribution of the HLA class II alleles DRB1 and DQB1 in 45 healthy volunteers and in 80 patients with paracoccidioidomycosis. The patients presented with various clinical forms of the disease, and allele distribution was evaluated individually in each presentation type. In patients with the unifocal chronic form of the disease, a mild clinical presentation in which lesions are restricted or localized, the HLA allele most commonly seen was DRB1*11 (p<0.039). This suggests that the participation of HLA antigens may influence the outcome of the host-parasite interaction in paracoccidioidomycosis, regulating the immune response to Paracoccidioides brasiliensis antigens.     

Early induction of MHC antigens in human liver grafts. An immunohistologic study.

The present study documents major histocompatibility complex (MHC) Class I and II expression during early acute rejection of human liver grafts. Serial graft biopsies (pretransplant, time zero, and 1 week) were studied. Ten patients received azathioprine (AZA) and prednisone; the other six patients were treated with quadruple therapy (azathioprine, cyclosporine A, prednisone, and cyclophosphamide). To study the specificity of changes in MHC antigen expression, biopsies of six patients with minor or no morphologic abnormalities served as controls. In addition, phenotypes of inflammatory cells present during rejection were analyzed using a panel of monoclonal antibodies. The results show that during acute rejection expression of MHC Class I and II antigens increased significantly in the AZA-treated patients, in a pattern similar to that seen in the patients treated with quadruple therapy, showing enhanced MHC Class I expression on hepatocytes, bile duct epithelium, and sinusoidal endothelium, and Class II antigen on Kupffer cells and sinusoidal endothelium. Bile duct epithelium was consistently positive for Class II antigen; no significant difference with the nonrejection group was observed. T cells are the predominant inflammatory cells during rejection with equal quantities of CD4+ and CD8+ cells. A majority of the infiltrating T cells show expression of Class II antigen but do not react with anti-interleukin-2 receptor antibody. This may be the result of immunosuppressive therapy or a simple reflection of the temporary expression of interleukin-2 receptors during lymphocyte activation. The authors hypothesize that the induction of MHC antigens on bile duct epithelium leads to rejection whereas the expression on hepatocytes represents an epiphenomenon.     

Progression of autoimmune damage in primary biliary cirrhosis: an immunohistochemical study

Aberrant MHC Class II antigen expression and the nature of the infiltrating lymphoid cells were studied by immunohistochemical techniques in liver biopsies from 37 patients with Primary biliary cirrhosis (PBC) (11 histological stage I, 13 stage II-III, 13 stage IV) and 15 patients with chronic non autoimmune liver disease. Bile duct epithelial cells expressed HLA-DR, DP and DQ antigens in biopsies from patients with early (Stage I) PBC and less frequently in the late cirrhotic phases of the disease (Stage IV); these observations support the hypothesis that induction of Class II antigens on epithelial cells may be involved in initiating autoimmune responses towards bile duct components. The presence of cytotoxic/suppressor T cells around the bile ducts in Stage I suggests a role for cell mediated destruction of the ducts at this early stage. The nature of the chronic inflammatory cell infiltrate in the portal tracts, periportal areas and lobular parenchyma does not establish the mechanism(s) involved in disease progression. However, the lack of Class II antigen expression on hepatocytes is compatible with the hypothesis that hepatocellular damage is non-specific and may be secondary to the initial bile duct injury.     

HLA expression on human germinal cells

Ejaculated human seminal plasma cells obtained from 46 healthy men and 48 infertile patients were tested for expression of HLA class I and II antigens by complement-mediated cytotoxicity, cell-binding radioimmunoassay (CB-RIA), and an ELISA involving monoclonal antibodies (mAbs). In a group of healthy men tested for HLA expression on human spermatozoa, 4 of 46 were positive for class I and II HLA antigens. However, the results were negative in a second examination of the same donors, possibly on account of alterations with time in the expression of various subpopulations of cells which are antigen positive or antigen negative. In a group of infertile patients, we found positive expression of HLA class I and II antigens in 7 of 48 men. The samples contained immature and mature sperm but no contaminating leucocytes or epithelial cells.     

Associated expression of HLA class I and class II antigens on melanoma cells in surgically removed metastases

Malignant transformation of melanocytes and further neoplastic progression may be associated with qualitative and/or quantitative changes in expression of HLA class I and class II antigens. Since previous immunohistochemical studies of surgically removed melanoma lesions have suggested a relationship in the expression of HLA class I and class II antigens, we have investigated the expression of these antigens at the single cell level. Double immunofluorescence staining of frozen sections of melanoma metastases and immunoelectron microscopic double labelling of melanoma cell suspensions prepared from three of these lesions has detected three HLA phenotypes on the large majority of melanoma cells: either both HLA class I and class II antigens, neither HLA antigen or only HLA class I antigens. In four out of the 11 lesions a few melanoma cells were found to express HLA class II antigens and to lack HLA class I antigens. A relationship was also found in the level of expression of HLA class I and class II antigens, as estimated by the intensity of staining with monoclonal antibodies. The level of expression of HLA class II antigens appeared to be similar to or lower than that of HLA class I antigens on the large majority of melanoma cells. This coordinated heterogeneity in the expression of HLA class I and class II antigens by melanoma cells may have implications in the interactions of tumour cells with the host's immune system.     

Expression of the Major Histocompatibility Complex Antigens on Different Liver Cellular Components in Rat and Man

We hint analysed the distribution of the major histocompatibility complex (MHC) antigens on different liver cellular components in rat and man. Two types of antisera were used: monospecific rabbit antisera raised against isolated molecules of class I and class II and ordinary hyperimmune alloantisera directed to their allelic specificities. A modified Staphylococcus aureus rosette test, enabling morphological identification of the rosette-forming cell type, was used. In both species the passenger cells, especially the Kupffer cells, reacted strongly with rabbit anti-class I and II The passenger erythrocytes reacted with anti-class I in rat but not in man. Human liver vascular endothelial and bile duct cells displayed a distinct reaction with anti-class I and II, whereas the hepatocytes reacted very weakly, if at all. In rat liver parenchymal component the expression of these antigens was different and weaker. Rat bile duct cells reacted with anti-class I and II in approximately 50% of the testings, whereas rat vascular endothelial cells and rat hepatocytes were essential non-reactive. The alloantiserum reactivity (tested only in the rat) followed closely that of heterologous antisera, and the reactivity of anti-β₂m that of class I alloantisera. Although these differences are probably quantitative rather than qualitative, the results suggest considerable variation in the expression of the MHC antigens on different liver cellular components. These differences partially explain why liver allografts behave differently than, e.g., renal allografts upon transplantation.     

The antigenic heterogeneity of the bile duct epithelium in alcoholic liver disease. VA Cooperative Study Group 275

The chronic alcoholic patient is usually immunosuppressed, but the significance of this phenomenon in terms of bile duct injury is unclear. The immunoreactivity of the bile duct cells was examined in a series of 69 frozen liver biopsy specimens obtained from patients with alcoholic liver disease, comprising 29 cases of cirrhosis, 26 of alcoholic hepatitis, 10 cases of alcoholic fatty liver, and 4 specimens from normal livers. Liver diseases such as primary biliary cirrhosis and human hepatic allograft rejection, known to have an autoimmune basis, share the characteristic feature of damage to the bile duct epithelial cells. In both instances the damage seems to be immune mediated, but the nature of the antigens involved is not established. We used the avidin-biotin-peroxidase complex method to test in alcoholic liver disease for the expression of a battery of surface antigen markers that have been incriminated in tissue injury and are usually present in lymphoid cells but also expressed by epithelium. In this study we investigated the expression of the following molecules: HLA class I (ABC) and class II (HLA-DR, HLA-DP, HLA-DQ), CD29, CD45RA, CD45RO, CD56, interleukin 1 (IL-I), IL-2, IL-4, interferon (IFN-gamma), tumor necrosis factor beta, and transforming growth factor beta1 (TGF-beta1). The bile duct epithelial cells strongly expressed HLA-ABC in all cases, CD56 in 47 of 55, IL-4 in 15 of 41, TGF-beta1 in 14 of 25, and CD29 in 4 of 25 cases. The other markers including IFN-gamma, HLA-DR, HLA-DP, and HLA-DQ were not expressed by bile duct cells. The expression of HLA class I agrees with previous observations while the absence of class II expression does not. The expression by the bile duct epithelium of CD56 confirms our own previous report. A new observation is the finding of molecules such as IL-4, TGF-beta1, and CD29 strongly expressed in the bile ducts cells. The presence of these molecules, taken together with the lack of IFN-gamma expression, contradicts previous speculations that attributed to IFN-gamma a role in the induction of major histocompatibility antigens and adhesion molecules in immune-mediated alcoholic liver disease.     

Class I and class II major histocompatibility complex antigens on hepatocytes: importance of the method of detection and expression in histologically normal and diseased livers

Methodological differences in major histocompatibility complex (MHC) antigen detection were investigated on isolated, viable hepatocytes and cryostat hepatic sections from 27 children with liver disorders, six of whom had normal histology. Class I antigens were constantly found on sections using a three step immunoperoxidase technique after acetone/chloroform fixation, other techniques being less sensitive, or on isolated hepatocytes by indirect immunofluorescence alone. With mechanical isolation the percentage of positivity ranged from 85 to 100%, while with collagenase isolation it ranged from 22 to 49% on immediate testing, and from 53 to 80% after 24 hour incubation. Class II antigens were only detected in one patient with autoimmune chronic active hepatitis and two with primary sclerosing cholangitis. Flow cytofluorimetric analysis in 11 cases confirmed class II or class I positivity, or both, on isolated hepatocytes, allowing MHC antigen expression on hepatocytes to be measured. Class I and II antigen detection on hepatocytes is influenced by the technique used. Although class I antigens are invariably expressed on hepatocytes, class II antigens are only found on hepatocytes from patients with immune mediated liver disorders.     

Loss of heterozygosity in the HLA class I region in human pancreatic cancer

An altered human leukocyte antigen (HLA) class I expression constitutes an important tumor-escape mechanism counteracting T-cell mediated immune responses. We utilized the technique of microsatellite analysis to characterize the loss of heterozygosity (LOH) in the HLA class I region in 24 samples of patients with human ductal pancreatic carcinoma. The expression of HLA class I and the infiltration by T cells were studied in parallel by standard immunohistochemistry. The present study demonstrates LOH in the HLA class I region in five patients with pancreatic carcinoma. Immunohistochemical analysis showed that pancreatic carcinomas were frequently characterized by a total or partial loss of expression in HLA class I antigen. The positive or negative LOH status corresponded with the expression analysis in eight cases. Reduction of HLA class I expression without LOH was found in 14 cases. Lymphocyte infiltration with CD3+, CD4+, and CD8+ T cells did not show significant differences between LOH-positive and LOH-negative tumors. In conclusion, LOH does not seem to be the only factor for the reduced expression of HLA class I antigen as well as for the T-cell infiltration in this type of tumor.     

HLA and TLX antigen expression on the human oocyte, zona pellucida and granulosa cells

The expression of human leukocyte antigen (HLA) class I and class II molecules and one trophoblast-lymphocyte crossreactive (TLX) antigen by human oocytes and granulosa cells was investigated. Well-defined monoclonal antibodies directed against HLA class I and class II molecules as well as beta 2-microglobulin and a TLX molecule were used in a standard indirect immunofluorescence test and some immunogold techniques at the electron microscopic level. Single unfertilizable or multiply fertilized oocytes and granulosa cells obtained from an in-vitro fertilization programme as well as oocytes in primary, secondary and tertiary follicles of ovaries were studied. Neither HLA class I or class II molecules, nor beta 2-microglobulin, nor a TLX molecule were detected on cultured oocytes or oocytes in human ovarian follicles or in their zona pellucida. Granulosa cells taken from a culture medium and those in follicles at various stages of development expressed class I antigens, while granulosa cells from tertiary follicles also expressed HLA-DR antigens. These results confirm that the female human gamete belongs to that very small group of cells that lack major histocompatibility complex antigens. Since spermatozoa also lack HLA antigens, human germ cells are entirely different from other nucleated human cells with regard to the antigenic structures expressed on the cell surface. This would prevent recognition of these cells by the cellular immune system. Furthermore, HLA and TLX antigens are not involved in fertilization and early differentiation.     

Altered pattern of major histocompatibility complex expression in renal carcinoma: tumor-specific expression of the nonclassical human leukocyte antigen-G molecule is restricted to clear cell carcinoma while up-regulation of other major histocompatibility complex antigens is primarily distributed in all subtypes of renal carcinoma

Renal epithelial cancers represent a heterogeneous group of neoplasms arising from the malignant transformation of presumed diverse cell lineages. We recently demonstrated that tumor-specific up-regulation of human leukocyte antigen (HLA)-G, a nonclassical HLA class Ib molecule that might be involved in immune evasion by tumor cells, frequently occurs in conventional (clear cell) renal carcinoma. We here examined whether HLA-G activation is a common process affecting all types of renal epithelial tumors. We analyzed a series of 38 paraffin-embedded tumors including clear cell, papillary, chromophobe, collecting duct carcinoma, and oncocytoma. Seven of 12 (58%) clear cell tumors were positive by immunohistochemistry, whereas all of the other subtypes of renal carcinoma were negative for HLA-G expression. Developing or adult normal renal tissue were devoid of HLA-G expression. We also observed that ectopic expression of HLA class II antigens occurs more frequently in clear cell renal carcinoma than in other subtypes of renal tumors. Moreover, in contrast to the common observation of a down-regulation of major histocompatibility complex class Ia antigens reported in various tumors, the concomitant study of the same biopsies for classical HLA class Ia antigen expression revealed a general increase of HLA class Ia expression, regardless of histological subtypes. These results provide evidence for the heterogeneity of major histocompatibility complex expression patterns in renal carcinoma and support the hypothesis that specific mechanisms underlying the malignant transformation into clear cell renal carcinoma up-regulate expression of HLA-G and to a lesser extent HLA class II molecule expression. Considering the immunotolerant role of HLA-G toward the immune response, these mechanisms may thus provide renal cell carcinoma tumor cells with additional means to escape immune surveillance.     

Altered expression of nonclassical HLA class Ib antigens in human renal cell carcinoma and its association with impaired immune response

An optimal antitumoral immune response requires the activation of both CD8(+) and CD4(+) T lymphocytes by the peptide antigen presentation via the human leukocyte antigen (HLA) class I and class II molecules, respectively. Downregulation or loss of HLA molecules has been found in human renal cell carcinoma (RCC) and provides a strategy of these tumors to evade T-cell mediated immunosurveillance. In addition, a tumor-specific upregulation of HLA-G has been recently described in RCC, which also leads to an impaired immune response. We here summarize the frequency of the constitutive and/or interferon-gamma (IFN-gamma) inducible expression of nonclassical HLA class Ib antigens in RCC cell lines, surgically removed RCC lesions and normal kidney epithelium, the molecular characteristics of HLA-G expression, and its role in immune recognition.     

Presence of Antibodies against Self Human Leukocyte Antigen Class II Molecules in Autoimmune Hepatitis

Autoimmune hepatitis (AIH) can arise de novo after liver transplantation (LT) for non-autoimmune liver diseases. Considering the identical features of de novo AIH after LT and classical AIH, as well as the importance of anti-human leukocyte antigen (HLA) antibodies in graft rejection, we investigated the presence of circulating anti-HLA class II antibodies in the sera of 35 patients with AIH, 30 patients with primary biliary cirrhosis (PBC), and 30 healthy donors using fluorescent dye-impregnated beads bound to HLA molecules. We then investigated the allele specificity of the antibodies and identified the HLA alleles in each patient using DNA-based HLA typing. We also examined HLA class II expression in liver samples using immunohistochemistry. Anti-HLA class II antibodies were detected significantly more frequently in the patients with AIH (88.1%) than in the patients with PBC (33.3%) or in the healthy donors (13.3%) (both P <0.01). We confirmed that the anti-HLA class II antibodies in the AIH patients showed specificity for several HLA class II alleles, including self HLA class II alleles. Moreover, positive reactivity with anti-self HLA class II antibodies was associated with higher serum transaminase levels. In conclusion, we demonstrated, for the first time, that antibodies against self HLA class II alleles were detectable in patients with AIH. Our results suggest that an antibody-mediated immune response against HLA class II molecules on hepatocytes may be involved in the pathogenesis or acceleration of liver injury in AIH.     

MHC class I and class II antigen expression in normal human corneas and in corneas from cases of herpetic keratitis

The expression of HLA class I and class II antigens in corneas from normal donors and patients with quiescent herpetic keratitis was investigated using specific monoclonal antibodies. Keratocytes from diseased corneas showed aberrant expression of HLA class I and class II (DR, DP and DQ) antigens. The expression of HLA antigens in these corneas was not associated with immune cell infiltrates or viral antigens.     

Reduced human leukocyte antigen expression in advanced‐stage Ewing sarcoma: implications for immune recognition

Ewing sarcoma (EWS) is a tumour most commonly arising in bone, although on occasion in soft tissue, with a poor prognosis in patients with refractory or relapsed disease, despite multimodal therapy. Immunotherapeutic strategies based on tumour‐reactive T and/or natural killer cells may improve the treatment of advanced‐stage EWS. Since cellular immune recognition critically depends on human leukocyte antigen (HLA) expression, knowledge about HLA expression in EWS is crucial in the design of cellular immunotherapeutic strategies. Constitutive and IFNγ‐induced HLA class I expression was analysed in EWS cell lines (n = 6) by flow cytometry, using antibodies against both monomorphic and allele‐specific antigens. Expression of antigen processing pathway components and beta‐2 microglobulin (β2m) was assessed by western blot. Expression of class II transactivator (CIITA), and its contribution to HLA class II expression, was evaluated by qRT‐PCR, transduction assays, and flow cytometry. β2m/HLA class I and class II expression was validated in EWS tumours (n = 67) by immunofluorescence. Complete or partial absence of HLA class I expression was observed in 79% of EWS tumours. Lung metastases consistently lacked HLA class I and sequential tumours demonstrated a tendency towards decreased expression upon disease progression. Together with absent or low constitutive expression levels of specific HLA class I loci and alleles, and differential induction of identical alleles by IFNγ in different cell lines, these results may reflect the existence of an immune escape mechanism. Inducible expression of TAP‐1/‐2, tapasin, LMP‐2/‐7, and the β2m/HLA class I complex by IFNγ suggests that regulatory mechanisms are mainly responsible for heterogeneity in constitutive class I expression. EWSs lack IFNγ‐inducible HLA class II, due to lack of functional CIITA. The majority of EWS tumours, particularly if advanced‐stage, exhibit complete or partial absence of both classes of HLA. This knowledge will be instrumental in the design of cellular immunotherapeutic strategies for advanced‐stage EWS. Copyright © 2009 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.