Down-regulation of the MHC class I antigen-processing machinery after oncogenic transformation of murine fibroblasts

Malignant transformation is often associated with genetic alterations providing tumor cells with mechanisms for escape from immune surveillance. Human and murine tumors of various origin as well as in vitro models of viral and oncogenic transformation express reduced levels of major histocompatibility complex (MHC) class I antigens resulting in decreased sensitivity to MHC class I-restricted cytotoxic T lymphocyte (CTL)-mediated lysis. We here investigate whether the suppressed MHC class I surface expression of ras-transformed fibroblasts is due to dysregulation of the genes of the antigen-processing machinery, the peptide transporters TAP-1 and TAP-2 and the proteasome subunits LMP-2 and LMP-7, and whether it can be restored by gene transfer. In comparison to parental NIH3T3 cells, the ras oncogenic transformants revealed reduced TAP and LMP mRNA expression and impaired function of these genes, leading to deficient peptide transport and peptide loading of MHC class I molecules resulting in instable expression of the MHC class I complex on the cell surface. Enhanced H-2 surface expression due to stabilization of the MHC class I complex could be achieved by culturing ras transformants at low, unphysiological temperature (26 °C) or by loading these cells with either exogenous human β2-microglobulin or MHC class I-binding peptide alone or in combination. Furthermore, interferon-γ treatment was capable to enhance the expression of TAP, LMP and MHC class I molecules in both parental as well as ras-transformed fibroblasts. Stable transfection of the human TAP-1 cDNA into ras transformants caused a partial reconstitution of the peptide transport and an enhancement of the MHC class I surface expression, whereas the level of MHC class I biosynthesis was not affected by TAP-1 overexpression in parental cells. Together these results point to the existence of an association between oncogenic transformation and deficiencies in the MHC class I antigen-restricted immunosurveillance, suggesting intervention strategies involving specific MHC class I-binding peptides or transfection of the LMP and/or TAP genes to overcome the expression of the immune escape phenotype.     

MHC class I immune evasion in MCMV infection

Murine cytomegalovirus (MCMV) is a well-studied model of natural β-herpesvirus infection. However, many questions remain regarding its control by and evasion of the immune response it generates. CD8 and CD4 T cells have both unique and redundant roles in control of the virus that differ based on the immunocompetence of the infected mice. MCMV encodes major histocompatibility complex (MHC) class I immune evasion genes that can have an impact in vitro, but their role in infection of immunocompetent mice has been difficult to identify. This review addresses the evidence for their in vivo function and suggests why they may be evolutionarily conserved.     

The murine gamma-herpesvirus-68 MK3 protein causes TAP degradation independent of MHC class I heavy chain degradation

The murine gamma-herpesvirus-68 MK3 protein has an intricate interaction with the peptide loading complex that involves MK3 stabilization, a rapid degradation of MHC class I heavy chains, and a slower degradation of TAP. Here we have used tapasin chimeras to distinguish functionally the different immune evasion mechanisms of MK3. Tapasin was cloned in two alternatively spliced forms that differed by a single transmembrane valine residue. Each restored antigen presentation and MK3 function in tapasin-deficient cells. The transmembrane/cytoplasmic portion of tapasin, linked to the extracellular domain of CD8, also restored TAP stability and MK3 stability in tapasin-deficient cells. MK3 did not associate with or degrade MHC class I in these cells, which lacked the endoplasmic reticulum domain of tapasin, but degraded TAP at least as efficiently as when full-length tapasin was present. The un-degraded MHC class I consequently showed impaired maturation. The fact that MK3 required intact tapasin to degrade MHC class I but only the transmembrane/cytoplasmic portion of tapasin to degrade TAP indicated that these two immune evasion functions operate independently.     

Activation of lymphocytes after platelet allotransfusion possessing only class I MHC product

After platelet allotransfusion, we found a characteristic increase in the expression of interleukin-2 receptor, dipeptydilpeptidase IV (CD26), activation-inducer molecule (AIM, CD69) and transferrin receptors (CD71) on day 3 indicating that important functional molecules expressed on the activation of lymphocytes by allogeneic platelets. At the same time, no consistent increase of other activation molecules such as Ki-l (CD30), intercellular adhesion molecule (ICAM-1, CD54) and Ki-24 (CDw70) antigen expression was detected, probably as a result of the selective activation of some lymphocyte subsets. In order to obtain further evidence for the in vivo activation triggered by allogeneic platelets, subsequent step of T cell activation towards differentiation was investigated with monoclonal antibodies to leucocyte common antigens. A sharp expression of the UCHL1, coupled with a decrease of the CD45R molecule was detected on day 7 or 14, suggesting a T cell priming.     

Phenotypic characterization of alveolar capillary endothelial cells,alveolar epithelial cells and alveolar macrophages in patients with pulmonary fibrosis,with special reference to MHC class II antigens

Summary Major histocompatibility complex (MHC) class II antigens are essential in the presentation of antigens to T lymphocytes, and cells expressing MHC class II antigens are known to play a role as antigen presenting cells (APC). We investigated the distribution of MHC class II antigens and the reactivity of monoclonal antibodies OKM1 and OKM5 in normal and fibrotic lungs immunohistochemically. The results showed that alveolar capillary endothelial cells (ACEnd) expressed MHC class II antigens and were reactive with OKM5 in both normal lungs and the non-thickened parts of alveolar septa of pulmonary fibrosis. However, ACEnd did not express MHC class II antigens and were not reactive with OKM5 in thickened alveolar septa of pulmonary fibrosis. Type II alveolar epithelial cells (AEp) proliferating and replacing type I AEp in pulmonary fibrosis expressed MHC class II antigens strongly. Alveolar macrophages expressed MHC class II antigens strongly and reacted with OKM1 in pulmonary fibrosis, especially in alveolar spaces. These findings suggest that the phenotypic changes of ACEnd may be involved in the process of pulmonary fibrosis, and type II AEp and alveolar macrophages in the parts of thickened alveolar septa may play a role as APC.     

Poor correspondence between predicted and experimental binding of peptides to class I MHC molecules

Naturally processed peptides presented by class I major histocompatibility complex (MHC) molecules display a characteristic allele specific motif of two or more essential amino acid side chains, the so-called peptide anchor residues, in the context of an 8-10 amino acid long peptide. Knowledge of the peptide binding motif of individual class I MHC molecules permits the selection of potential peptide antigens from proteins of infectious organisms that could induce protective T-cell-mediated immunity. Several methods have been developed for the prediction of potential class I MHC binding peptides. One is based on a simple scanning for the presence of primary peptide anchor residues in the sequence of interest. A more sophisticated technology is the utilization of predictive computer algorithms. Here, we have analyzed the experimental binding of 84 peptides selected on the basis of the presence of peptide binding motifs for individual class I MHC molecules. The actual binding was compared with the results obtained when analyzing the same peptides by two well-known, publicly available computer algorithms. We conclude that there is no strong correlation between actual and predicted binding when using predictive computer algorithms. Furthermore, we found a high number of false-negatives when using a predictive algorithm compared to simple scanning for the presence of primary anchor residues. We conclude that the peptide binding assay remains an important step in the identification of cytotoxic T lymphocyte (CTL) epitopes which can not be substituted by predictive algorithms.     

NK receptor interactions with MHC class I molecules in pregnancy

Both HLA class I molecules and their receptors on Natural Killer cells, the KIR molecules, are highly polymorphic. It is generally believed that this variation is driven in response to the role of these receptors and counter-receptors in resistance to disease. Uterine NK cells are the major maternal leukocyte population present within the decidua, and they express KIR2D receptors for HLA-C, the only polymorphic class I molecule on trophoblast. Genetic and functional data suggest that the maternal KIR/fetal HLA-C interaction in pregnancy may affect the delivery of an optimal blood supply to mother and fetus. The drive for novelty in HLA-C and KIR2D allelic diversity may relate not only to survival from infections but also to reproductive success.     

Fc receptors for IgG and antigen presentation on MHC class I and class II molecules

Antigens internalized through specific membrane receptors are presented to helper CD4(+) T cells at antigen concentrations 10(3) to 10(4) fold lower than antigens internalized by fluid phase. B lymphocyte antigen receptors, mannose receptors and receptors for the Fc region of immunoglobulins, promote both internalization and efficient presentation at low antigen concentrations. Thus, binding to specific membrane receptors concentrate antigens on antigen presenting cells and mediates efficient uptake. Is this 'quantitative' concentration of antigens on antigen presenting cells the end of the story? Or may 'quality', i.e. selective intracellular antigen targeting, somehow influence the efficiency or specificity of MHC class I and class II-restricted antigen presentation?     

Induction of antigen-presenting capacity in tumor cells upon infection with non-replicating recombinant vaccinia virus encoding murine MHC class II and costimulatory molecules

The possibility of inducing antigen-presenting capacity in cells normally lacking such capacity, currently represents a major goal in vaccine research. To address this issue we attempted to generate ‘artificial’ APC able to stimulate CD4⁺ T cell responses when tumor cells were infected with a single, recombinant, vaccinia virus (rVV) containing the two genes encoding murine MHC class II I-A^k and a third gene encoding the murine B7-1 (mB7-1) costimulatory molecule. To minimize the cytopathic effect and to improve safety, in view of possible in vivo applications, we made this rVV replication incompetent by Psoralen and long wave UV treatment. Tumor cells infected with rVV encoding I-A^k alone, pulsed with hen egg white lysozyme peptide (HEL_46–61), induced IL-2 secretion by an antigen-specific T hybridoma. Tumor cells infected with the rVV encoding mB7-1 provided costimulation for activating resting CD4⁺ T cells in the presence of ConA. Tumor cells infected with the rVV encoding I-A^k and mB7-1, and pulsed with chicken ovotransferrin peptide (conalbumin_133–145), induced a significantly higher response in a specific Th₂ cell clone (D10.G4.1) as compared to cells infected with rVV encoding I-A^k molecules only. Thus, this replication incompetent rVV represents a safe, multiple gene, vector system able to confer in one single infection step effective APC capacity to non-professional APCs.     

Localization of MHC class II molecules in murine bone marrow-derived macrophages.

The localization of MHC class II molecules (Ia) was studied by ultrastructural immunocytochemistry in murine bone marrow-derived macrophages stimulated by recombinant interferon-gamma (rIFN-gamma). Ia molecules were detected on the plasma membrane and on the limiting membrane and internal structures of vesicular acidic compartments. Some of these vesicules also contained cathepsin B and/or cathepsin D. The use of BSA-gold, a marker of fluid phase endocytosis, allowed the identification of Ia-positive organelles as endocytic compartments. The first to be labelled with BSA-gold also contained the cation-independent mannose 6-phosphate receptor (MPR) but not the 120,000 molecular weights lysosomal glycoprotein (lgp 120). Later on, BSA-gold appeared in Ia+, MPR+, lgp 120+ compartments. Collectively these data suggest that intracellular Ia molecules are mainly present in early and late endosomes.     

Blocking oncogenic RAS enhances tumour cell surface MHC class I expression but does not alter susceptibility to cytotoxic lymphocytes

Mutations in the RAS family of oncogenes are highly prevalent in human cancer and, amongst its manifold effects, oncogenic RAS impairs the expression of components of the antigen presentation pathway. This allows evasion of cytotoxic T lymphocytes (CTL). CTL and natural killer (NK) cells are reciprocally regulated by MHC class I molecules and any gain in CTL recognition obtained by therapeutic inactivation of oncogenic RAS may be offset by reduced NK cell activation. We have investigated the consequences of targeted inactivation of oncogenic RAS on the recognition by both CTL and NK cells. Inactivation of oncogenic RAS, either by genetic deletion or inactivation with an inducible intracellular domain antibody (iDAb), increased MHC class I expression in human colorectal cell lines. The common RAS mutations, at codons 12, 13 and 61, all inhibited antigen presentation. Although MHC class I modulates the activity of both CTL and NK cells, the enhanced MHC class I expression resulting from inactivation of mutant KRAS did not significantly affect the in vitro recognition of these cell lines by either class of cytotoxic lymphocyte. These results show that oncogenic RAS and its downstream signalling pathways modulate the antigen presentation pathway and that this inhibition is reversible. However, the magnitude of these effects was not sufficient to alter the in vitro recognition of tumour cell lines by either CTL or NK cells.     

LAMP-2-deficient human B cells exhibit altered MHC class II presentation of exogenous antigens

Major histocompatibility complex (MHC) class II molecules present antigenic peptides derived from engulfed exogenous proteins to CD4⁺ T cells. Exogenous antigens are processed in mature endosomes and lysosomes where acidic proteases reside and peptide‐binding to class II alleles is favoured. Hence, maintenance of the microenvironment within these organelles is probably central to efficient MHC class II‐mediated antigen presentation. Lysosome‐associated membrane proteins such as LAMP‐2 reside in mature endosomes and lysosomes, yet their role in exogenous antigen presentation pathways remains untested. In this study, human B cells lacking LAMP‐2 were examined for changes in MHC class II‐restricted antigen presentation. MHC class II presentation of exogenous antigen and peptides to CD4⁺ T cells was impaired in the LAMP‐2‐deficient B cells. Peptide‐binding to MHC class II on LAMP‐2‐deficient B cells was reduced at physiological pH compared with wild‐type cells. However, peptide‐binding and class II‐restricted antigen presentation were restored by incubation of LAMP‐2‐negative B cells at acidic pH, suggesting that efficient loading of exogenous epitopes by MHC class II molecules is dependent upon LAMP‐2 expression in B cells. Interestingly, class II presentation of an epitope derived from an endogenous transmembrane protein was detected using LAMP‐2‐deficient B cells. Consequently, LAMP‐2 may control the repertoire of peptides displayed by MHC class II molecules on B cells and influence the balance between endogenous and exogenous antigen presentation.     

Hepatitis B Virus Down-Regulates Expressions of MHC Class Ⅰ Molecules on Hepatoplastoma Cell Line

Chronic HBV infection is associated with a 100-fold high risk of developing hepatocellular carcinoma. Tumor recognition is of the most importance during the immune surveillance process that prevents cancer development in humans. In the present study, the expressions of MHC class Ⅰ molecules on hepatoplastoma cell line HepG2.2.15 were investigated to indicate the possible effects of HBV on the immune recognition during HBV-associated hepatocellular carcinoma. It was found that the expressions of MHC class Ⅰ molecules HLA-ABC, HLA-E and MICA were much lower in HepG2.2.15 cells compared with HepG2 cells. The expressing HBV in human hepatoplastoma cell line significantly down-regulated the expressions of MHC class Ⅰ molecules. Additionally, it was observed that in murine chronic HBsAg carriers the expression of classical MHC-I molecule on hepatocytes was down-regulated. These results demonstrated that HBV might affect the immune recognition during HBV- associated hepatocellular carcinoma such as the recognition of CD8^＋ T, NK-CTL and NK cells and prevent the immune surveillance against tumors. However, the effects of HBV down-regulation of MHC class I molecules on the target cells in vivo should be further studied. Cellular ＆ Molecular Immunology.     

Interferon-induced expression of class II major histocompatibility antigens in the major histocompatibility complex (MHC) class II deficiency syndrome

Class II antigens encoded by genes of the major histocompatibility complex (MHC) are expressed by a variety of cell types and have a vital role in the cellular interactions required for an effective immune response. We have analyzed the regulation of HLA-DR, DP, and DQ class II antigen expression on cells of different lineage from an immunodeficient patient with the MHC class II deficiency syndrome. T and B lymphocytes, monocytes, and fibroblasts, which initially expressed no class II antigens, were treated with inductive stimuli that normally lead to enhanced expression of class II antigens. Monocytes, but not fibroblasts, cultured for 48–96 hr in the presence of recombinant gamma interferon expressed all three types of class II antigens. In contrast, T lymphocytes did not express class II antigens following their exposure to a variety of stimuli, including activation with phytohemagglutinin and culture in the presence of interleukin-2, transformation by the retrovirus HTLV-1 or HTLV-2, or exposure to the demethylating agent 5-azacytidine. Similarly, class II antigens were not induced on B cells by cross-linkage of surface immunoglobulin molecules with anti-mu, exposure to Epstein-Barr virus, or treatment with soluble factors secreted by activated T cells. These results demonstrate that the regulation of class II MHC antigen expression by monocytes and lymphocytes is dissimilar and suggest that different regulatory genes are involved in the control of class II antigen expression by cells of different lineage.     

Abnormal differentiation of immunoregulatory T-lymphocyte subpopulations in the major histocompatibility complex (MHC) class II antigen deficiency syndrome

The major histocompatibility complex (MHC) class II deficiency syndrome is a rare immunodeficiency disease associated with defective expression of class II MHC antigens. We have examined the consequences of this defect for the differentiation and functional capabilities of immunoregulatory T-cell subpopulations in an affected patient. Although the number of circulating T cells was normal, there was a striking reduction in the number of CD4+ T cells. Furthermore, purified CD4+ cells from the patient were unable to provide help for antibody secretion. This defect in helper function appeared to be due to the abnormal differentiation of the few CD4+ cells present, virtually all of which expressed the CD4 + HB11 + phenotype characteristic of immature virgin T cells. Abnormal development of immunoregulatory CD8+ T cells was also observed. Although increased numbers of CD8+ T cells were present, virtually none had phenotypic properties of suppressor cells (i.e., CD3+/CD8+/9.3-granular lymphocytes that coexpress the Leu-15 or Leu-7 antigens), and purified CD8+ cells from the patient had no suppressor activity. Thus, the absence of class II MHC antigens profoundly disrupts the development of immunoregulatory T cells. We propose that these effects occur by the following mechanisms: (1) the absence of intrathymic class II antigens results in deficient production of CD4+ cells, (2) the CD4+ cells that do emerge from the thymus do not undergo postthymic maturation into CD4+HB11- cells with helper capabilities, and (3) the absence of CD4+HB11- effector cells results in abortive development of suppressor cells involved in feedback suppression.     

MHC class II antigen presentation and immunological abnormalities due to deficiency of MHC class II and its associated genes

Antigen presentation by Major Histocompatibility Complex (MHC) class II molecules plays an important role in controlling immunity and autoimmunity. Multiple co-factors including the invariant chain (Ii), HLA-DM and HLA-DO are involved in this process. While the role for Ii and DM has been well defined, the biological function of DO remains obscure. Our data indicate that DO inhibits presentation of endogenous self-antigens and that developmentally-regulated DO expression enables antigen presenting cells to preferentially present different sources of peptide antigens at different stages of development. Disruption of this regulatory mechanism can result in not only immunodeficiency but also autoimmunity. Despite the fact that deletion of each of the three genes in experimental animals is associated with profound immunological abnormalities, no corresponding human diseases have been reported. This discrepancy suggests the possibility that primary immunodeficiencies due to a genetic defect of Ii, DM and DO in humans are under diagnosed or diagnosed as “common variable immunodeficiency”, a category of immunodeficiency of heterogeneous or undefined etiology. Clinical tests for any of these potential genetic defects are not yet available. We propose the use of multi-color flow cytometry in conjunction with intracellular staining to detect expression of Ii, DM, DO in peripheral blood B cells as a convenient reliable screening test to identify individuals with defects in antigen presentation.     

MHC class II molecules activate NFAT and the ERK group of MAPK through distinct signaling pathways in B cells

MHC class II (MHC‐II) molecules are capable of transducing signals with the help of associated molecules. Although the search to find associated molecules over the past few years has been fruitful, it remains clear that not all signaling components and their mechanisms of action have been identified. In this study, we investigated calcium and MAPK signaling pathways using the BJAB and Raji human B cell lines. We demonstrate that calcium mobilization is an isotype‐independent event that triggers the dephosphorylation of NFAT. We also show that BCR activation followed by MHC‐II ligation increases the activation of NFAT. This signaling pathway differs from MHC‐II‐mediated MAP activation, where MEK1/2 and ERK1/2 phosphorylation are isotype‐specific events, which correspond to the induction of c‐Fos and formation of AP‐1. Future studies should elucidate the intertwined, intricate signaling cascades triggered by BCR and MHC‐II leading to humoral immune responses.     

Down-regulation of MHC class I is a property common to papillomavirus E5 proteins

The E5 protein family of papillomaviruses comprises small hydrophobic proteins which are associated with the cell endomembrane compartments. The functions of the E5 proteins, particularly those of HPV, are still far from clear. We have reported that the E5 proteins of BPV-1, BPV-4, HPV-16 and HPV-6 down-regulate MHC class I, potentially helping the virus evade the host immune response. Others have described MHC class I down-regulation by HPV-2 E5. We report here that another E5 protein, HPV-83 E5, likewise down-regulates MHC class I and propose that interference with expression, assembly and/or transport of MHC class I is a common property of all E5 proteins evolved by the virus to circumvent host immunosurveillance and thus establish productive infection.     

High‐resolution analysis of the murine MHC class II immunopeptidome

The reliable identification of peptides bound to major histocompatibility complex (MHC) class II is fundamental for the study of the host immune response against pathogens and the pathogenesis of autoimmune conditions. Here, we describe an improved methodology combining immuno‐affinity enrichment of MHC class II complexes, optimized elution conditions and quadrupole Orbitrap mass spectrometry‐based characterization of the immunopeptidome. The methodology allowed the identification of over 1000 peptides with 1% false discovery rate from 10⁸ murine A20 lymphoma cells. The study revealed the I‐A^d‐specific motif in high resolution after multisequence alignment. The methodology was generally applied to the purification of MHC class II from cell lines and murine spleens. We identified 2963 peptides from BALB/c and 2712 from C57BL/6 mouse spleens. The identification of peptides bound to MHC class II in vitro and in vivo will facilitate the characterization of T‐cell specificities, as well as the development of biotherapeutics and vaccines.