Antigen processing mutant T2 cells present viral antigen restricted through H-2K

Cytotoxic T lymphocytes (CTL) recognize foreign antigens as short peptides presented by class I molecules of the major histocompatibility complex (MHC). T2 cells are profoundly defective in the presentation of endogenously synthesized antigens to CTL due to a deletion of MHC class II-encoded genes for transporters associated with antigen presentation (TAP1/TAP2). Surprisingly, we here demonstrate that T2 cells, after infection with Sendai virus, are readily killed by H-2K^b restricted CD8⁺ T cells. In contrast to classical class I-mediated antigen presentation, the presentation of Sendai virus antigen inT2K^b cells is brefeldin A (BFA) insensitive. The present findings may suggest the presence of an alternative pathway for MHC class I-mediated antigen presentation in T2 cells.     

Genes encoded in the major histocompatibility complex affecting the generation of peptides for TAP transport

The B cell line 721.174 has lost the ability to present intracellular antigens to major histocompatibility complex (MHC) class I-restricted cytotoxic T lymphocytes (CTL). This phenotype results from a homozygous deletion in the MHC that includes the peptide transporter genes TAP1 and TAP2, and the proteasome subunits LMP2 and LMP7. Recent work has shown that such cells transfected with TAP genes load their class I molecules with endogenous peptides, and present several viral epitopes to class I-restricted CTL. These data implied that the LMP2 and LMP7 genes were not required for the presentation of most epitopes through class I molecules. By contrast, while confirming the previous reports, we have identified several epitopes that appear to require genes in the MHC in addition to the TAP for their presentation. Further analysis localizes the defect to proteolysis in the cytosol. In one case, presentation could be partially restored by re-expression of full-length LMP7. Control experiments with LMP7, from which the putative pro-region had been removed, failed to restore presentation, and this lack of effect correlated with failure of the shortened LMP7 to incorporate into the proteasome. These results suggest a role for LMP7 in the generation of a viral epitope, but leave open the possibility that additional genes within the .174 deletion are required for full restoration of antigen presentation.     

Characterization of TAP-Independent and Brefeldin A-Resistant Presentation of Sendai Virus Antigen to CD8+ Cytotoxic T Lymphocytes

H-2K^b-transfected T2 cells, which lack both TAP1/2 and LMP2/7 genes, are able to efficiently process and present Sendai virus Antigen to K^b-restricted Sendai virus-specific CTL. This presentation is not inhibited by Brefeldin A (BFA). Here we extend our analysis of this novel antigen presentation pathway. We show that presentation of Sendai virus antigen was not due to sensitization of T2K^b cells by peptides in the virus preparation or peptides released from virus infected cells. Also, the ability to present Sendai virus in a BFA resistant fashion was specific for cells of the T2 lineage. Re-expression of TAP1/2 genes in T2K^b cells did not alter the capability to present antigen in a BFA resistant fashion, i. e. the presence of a functional TAP transporter complex did not relocate (all) peptides to the classical pathway for antigen processing and presentation. We found that co-infection of T2K^b cells with either Sendai virus plus influenza virus or Sendai virus plus VSV did not relocate presentation of influenza or VSV antigen to the TAP independent BFA resistant antigen presentation pathway. Peptide elution experiments and studies with peptide-specific CTL firmly demonstrated that the antigen presented by T2K^b cells after infection with Sendai virus was the natural Sendai virus epitope NP324-332. The same epitope, when expressed as a minigene in vaccinia virus, could be presented also by T2K^b cells but this presentation could be blocked by BFA. Thus, the TAP independent BFA resistant presentation of antigen seem cell (T2 lineage) and virus (Sendai virus) specific, but not epitope specific. The ability of T2K^b cells to present Sendai virus antigen in a TAP independent BFA resistant fashion was only partially blocked by lysosomal inhibitors such as methylamine, ammonium chloride and chloroquine. These findings demonstrate that TAP1/2-independent and BFA-resistant class I processing is only expressed in certain cell types, in parallel with classical MHC class I processing, and that Sendai virus selectively can enter this pathway. Hypothetical models for the TAP-independent class I processing are discussed.     

Markedly decreased expression of TAP1 and LMP2 genes in HLA class I-deficient human tumor cell lines

HLA class I antigens of the human major histocompatibility complex play an important role in immune response. These molecules present foreign antigenic peptides to cytotoxic T lymphocytes and thereby play a role in the immune surveillance of cells infected with virus or other intracellular pathogens or altered by malignant transformation. A marked deficiency or lack of expression of these antigens has been reported in a variety of human neoplasms. In the present study, we examined the expression of class I chain, β₂-microglobulin, TAP (TAP1 and TAP2) and LMP (LMP2 and LMP7) genes in a number of human tumor cell lines including small-cell lung carcinoma, hepatocellular carcinoma, colon adenocarcinoma and basophilic leukaemia. These cell lines were deficient in expression of both class I chain and β₂-microglobulin gene products. In addition, these cell lines lacked the products of MHC-encoded proteasome subunit LMP2 as well as the putative peptide transporter TAP1 genes. In contrast, TAP2 and LMP7 genes were expressed in these cell lines. Treatment of cells with γ-IFN markedly enhanced the expression of class I chain, β₂-microglobulin, TAP1 and LMP2 genes with a concomitant increase in cell-surface expression of class I molecules. The upregulation of TAP1 and LMP2 expression is associated with increased class I expression, suggesting that endogenous antigens, e.g. tumor antigens, could be presented by class I molecules following treatment of tumor cells with γ-IFN.     

Genes of the LMP/TAP cluster are associated with the human autoimmune disease vitiligo

Genes within the class II region of the major histocompatibility complex (MHC), including genes involved in antigen processing and presentation, have been reported to be associated with several autoimmune diseases. We report here that the LMP/TAP gene region is significantly associated with vitiligo, a disorder in which biochemical defects and/or autoimmune destruction cause melanocyte loss and resulting skin depigmentation. Case/control analyses revealed genetic association of vitiligo in Caucasian patients with an early age of onset with the transporter associated with antigen processing-1 (TAP1) gene. A family-based association method revealed biased transmission of specific alleles from heterozygous parents to affected offspring for the TAP1 gene, as well as for the closely linked LMP2 and LMP7 genes encoding subunits of the immunoproteasome. No association with vitiligo was found for the MECL1 gene, which encodes a third immunoproteasome subunit and is unlinked to the MHC class II region. These results suggest a possible role for the MHC class I antigen processing and/or presentation pathway in the antimelanocyte autoimmune response involved in vitiligo pathogenesis.     

Polymorphism of LMP2, TAP1, LMP7 and TAP2 in Brazilian Amerindians and Caucasoids: implications for the evolution of allelic and haplotypic diversity

In the class II region of the major histocompatibility complex (MHC), four genes implicated in processing of MHC class I‐presented antigens have been described. Two of these (TAP1 and TAP2) code for endoplasmic reticulum membrane transporter proteins and the other two (LMP2 and LMP7) for proteasome subunits. These genes are polymorphic, although much less so than classical MHC class I and II genes. There is controversy concerning the possible functional implications of this variation. Population genetics is one of the means of investigating the evolutionary and functional significance of genetic polymorphisms; however, few populations have been analysed with respect to TAP and LMP diversity. We present here the polymorphism of TAP1, TAP2, LMP2 and LMP7 genes in the Kaingang and Guarani Amerindian tribes, and in the Caucasoid population of the Brazilian State of Paraná. Allele frequencies found in the Caucasoids were close to those described for similar populations. Amerindians had a somewhat more restricted polymorphism, and allele and haplotype frequencies differed greatly between the two tribes. Overall linkage disequilibrium (LD) between the four genes was low in the Caucasoids, but high in the Amerindians, for which significant LD was seen for all informative pairs of loci. Comparing results of this and previous studies we observed that, whenever significant LD occurs in non‐Amerindians, it tends to be similar in the different ethnic groups. While this might be interpreted as evidence of co‐evolution of genes in the TAP‐LMP region, the high haplotypic diversity in all populations and low LD in non‐Amerindians indicate absence of co‐evolution of the different genes. Distributions of allele and genotype frequencies are consistent with the hypothesis of selective neutrality. We conclude that genetic polymorphism of the human TAP and LMP genes and haplotypes is of little, if any, functional significance.     

MHC Class I Presentation of Live and Heat-Inactivated Sendai Virus Antigen in T2Kb Cells Depends on an Intracellular Compartment with Endosomal Characteristics

T2K^b cells, which do not express TAP1/2 peptide transporters or the low molecular weight protein 2/7 (LMP2/7) proteasomal subunits, can still process and present both live and heat-inactivated Sendai virus (SV). As this operation may also reflect the existence of an alternative processing pathway in normal antigen-presenting cells (APC), the authors have characterized it using intracelluular inhibitors and anti-K^b monoclonal antibodies (MoAbs). From the results with lipophilic amines (ammonium chloride, methylamine and chloroquine), cytoskeletal inhibitors (cytochalasin B and vinblastine), and an endoprotease inhibitor (phenylmethylsulfonyl fluoride, PMSF), the authors conclude that the processing of SV antigen in T2K^b cells has endosomal characteristics depending on cellular activities such as uptake, vesicular transport and intracellular–vesicular proteolysis. In addition, internalized ‘empty’ K^b molecules derived from the T2K^b cell surface appeared to be involved in the presentation of SV antigen, as demonstrated by a protocol using a combination of the Golgi inhibitor brefeldin A(BFA) and anti-K^b antibodies. The results thus indicate that T2K^b cells process SV antigen in an endosomal-like compartment which contain recycling ‘empty’ K^b molecules.     

Differential effect of transporter Tap 2 gene introduction into RMA-S cells on viral antigen processing

The protein products of the Tap (Transporter associated with antigen processing) 1 and 2 genes are presumed to deliver peptides across the endoplasmic reticulum (ER) for assembly with major histocompatibility complex (MHC) class I molecules. The antigen processing-defective cell line RMA-S (H-2^b) has a premature stop in the Tap 2 gene and probably therefore fails to deliver peptides into the ER, which leads to a low level of cell surface MHC class I molecules. Transfection of a Tap 2 gene restores to RMA-S both MHC class I molecule expression and the ability to present influenza viral antigens. We investigated the ability of RMA-S cells transfected with a Tap 2 gene to process and present alloantigens, Sendai and Rauscher viral antigens to allogeneic and virus-specific cytotoxic T lymphocytes. We found that allogeneic peptides as well as Rauscher and Sendai viral peptides can be processed and presented by RMA-S but at reduced levels. Transfection of a Tap 2 gene of mouse (BALB/c, H-2^d) or rat origin into RMA-S increased the presentation of Sendai viral antigens and partially restored the presentation of allogeneic antigens. The already low level of Rauscher viral peptides presented by RMA-S is not elevated by transfection of either Tap 2 gene into RMA-S. This indicates a differential effect of transfection of a Tap 2 gene of rat or allogeneic mouse origin into RMA-S on viral antigen processing.     

Critical role for the immunoproteasome subunit LMP7 in the resistance of mice to Toxoplasma gondii infection

Proteasome‐mediated proteolysis is responsible for the generation of immunogenic epitopes presented by MHC class I molecules, which activate antigen‐specific CD8⁺ T cells. Immunoproteasomes, defined by the presence of the three catalytic subunits LMP2, MECL‐1, and LMP7, have been hypothesized to optimize MHC class I antigen processing. In this study, we demonstrate that the infection of mice with a protozoan parasite, Toxoplasma gondii, induced the expression of LMP7 mRNA in APC and increased the capacity of APC to induce the production of IFN‐γ by antigen‐specific CD8⁺ T cells. In vitro infection of a DC cell line with T. gondii also induced the expression of LMP7 and resulted in enhanced proteasome proteolytic activity. Finally, mice lacking LMP7 were highly susceptible to infection with T. gondii and showed a reduced number of functional CD8⁺ T cells. These results demonstrate that proteasomes containing LMP7 play an indispensable role in the survival of mice infected with T. gondii, presumably due to the efficient generation of CTL epitopes required for the functional development of CD8⁺ T cells.     

Anti-viral immune responses in a primitive lung: Characterization and expression analysis of interferon-inducible immunoproteasome subunits LMP2, LMP7 and MECL-1 in a sarcopterygian fish,the Nigerian spotted lungfish (Protopterus dolloi)

Lungfishes (Dipnoi) represent the closest ancestor of tetrapods. Dipnoi have dual breathing modes extracting oxygen from water and air. The primitive lungs of lungfishes are exposed to external antigens including viruses. To date, the immune response of lungfishes against viruses has not been investigated. During viral immune responses, cell exposure to type I interferon induces the replacement of the constitutive proteasome with LMP2, LMP7 and MECL-1 beta subunits forming the immunoproteasome and enhancing antigen presentation to MHC class I molecules. In order to study the immune defense system of the lungfish lung, we have characterized for the first time the three immunoproteasome subunits in the sarcopterygian fish, the Nigerian spotted lungfish (Protopterus dolloi). LMP2, LMP7 and MECL-1 were identified in P. dolloi and their sequences encoded predicted proteins of 216, 275 and 278 amino acids, respectively. The mRNA of these three genes was expressed in multiple tissues, including the lung, with the highest abundance observed in kidney and post-pyloric spleen. In vitro stimulation of lungfish lung and kidney primary cell cultures with PolyI:C for 4 and 12 h resulted in increased LMP2, LMP7 and MECL-1 expression in both tissues. These results suggest a central role of these genes in the activation of an antiviral immune response in lungfish. Importantly, they indicate that the primitive lung of the common ancestor of all tetrapods is capable of inducing the expression of these genes in response to viral stimulation.     

Characterization of LMP polymorphism in homozygous typing cells and a random population

Within the class II region of the MHC are several genes whose products are involved in processing antigen for HLA class I presentation. Two such genes, LMP2 and LMP7, encode products that are incorporated into a multicatalytic proteinase complex which serves as the major pathway for protein degradation for class I peptide presentation. Polymorphic residues have been identified in both LMP2 and LMP7. In this report, we describe an ARMS-PCR method to distinguish LMP7 alleles. We applied this method to characterize these alleles in addition to LMP2 alleles in 50 homozygous typing cells (HTC) as well as in a panel of 110 random individuals. Of the four possible combinations of LMP2 and LMP7, we observed three in the HTC population, while all four were observed in the random population. The frequencies at which allele combinations were observed were similar to that predicted by individual allele frequencies. We also analyzed the possibility of linkage disequilibrium of LMP2 and LMP7 alleles with TAP1, TAP2, and specific HLA class I alleles in both populations. From this data, there seems to be no apparent linkage disequilibrium and no indication that particular combinations of LMP2 and LMP7 have been maintained.     

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.     

Development and characterization of mouse anti-human LMP2, LMP7, TAP1 and TAP2 monoclonal antibodies

Abstract: Low molecular mass polypeptides (LMP) 2 and LMP7 and transporter associated with antigen processing (TAP) subunits TAP1 and TAP2 play a crucial role in antigen processing and cell surface expression of HLA class I molecules. Since monoclonal antibodies (mAb) to these molecules will facilitate the analysis of their expression, structure and function in normal and transformed cells, in the present study we have developed these reagents. Specifically anti-LMP2 and LMP7 mAb were generated from BALB/c mice immunized with specific peptides, and anti-TAPl and TAP2 mAb from BALB/c mice immunized with respective recombinant proteins. mAb VF101–39F7 and VF1O1–39G5 were shown to be specific for LMP2, mAb VF103–5D5 and VF103–8C2 for LMP7, mAb VF108–1B3 and VF108–12D6 for TAP1 and mAb VF118–1E4 and VF118–2C5 for TAP2, since they reacted specifically with the corresponding immunogens in ELISA and with the corresponding LMP and TAP subunits when tested in Western blotting with human lymphoid cell extracts. Furthermore, the mAb immunoprecipitated components with the characteristic electrophoretic mobility from lymphoid cells. Both anti-LMP and anti-TAP mAb stained keratinocytes and infiltrating lymphocytes in frozen and formalin-fixed, paraffin embedded sections of normal skin in indirect immunoperoxidase reactions. Furthermore, all the mAb except mAb VF103–5D5 stained the cytoplasm of lymphoid cells in an intracytoplasmic staining reaction. The specificity and reactivity pattern of the mAb we have characterized indicate that they will be valuable reagents to analyze the cellular expression and tissue distribution of LMP and TAP subunits.     

A third interferon-γ-induced subunit exchange in the 20S proteasome

The 20S proteasome is a protease complex of functional importance for antigen processing. Two of the 14 proteasome subunits, δ and MB1, can be replaced by the major histocompatibility complex (MHC)-encoded and interferon-γ (IFN–γ)-inducible subunits LMP2 and LMP7, respectively. LMP2 and LMP7 alter the cleavage site specificity of the 20S proteasome and are required for the efficient generation of T cell epitopes from a number of viral proteins and for optimal MHC class I cell surface expression. We compared the 20S proteasome subunit pattern from IFN-γ-induced and non-induced mouse fibroblasts on two-dimensional gels and identified a third subunit exchange by microsequencing: the non-MHC-encoded subunit MECL-1 is induced by IFN-γ and replaces a so-far barely characterized β subunit designated ‘MC14’. In analogy to LMP2 and LMP7, MECL-1 may be functional in MHC class I-restricted antigen presentation.     

Down-regulation of HLA class I antigen-processing molecules in malignant melanoma: association with disease progression

Expression of the proteasome subunits LMP2 and LMP7, the MHC-encoded transporter subunits TAP1 and TAP2, and HLA Class I antigens was examined by immunoperoxidase staining in 10 nevi and 98 melanoma lesions (60 primary and 38 metastatic), because these molecules play an important role in the presentation of melanoma-associated peptide antigens to cytotoxic T cells. LMP2 was less frequently expressed than LMP7 in primary and metastatic melanoma lesions. TAP1, TAP2, and HLA Class I antigen expression was more frequently (P < 0.05) down-regulated in metastatic than in primary melanoma lesions and in nevi. A synchronous TAP1, TAP2, and HLA Class I antigen down-regulation was observed in 58% of primary and 52% of metastatic lesions. TAP and HLA Class I antigen down-regulation in primary lesions was significantly associated with lesion thickness, stage of disease, reduced time to disease progression, and reduced survival. These results suggest that TAP down-regulation plays a role in the clinical course of malignant melanoma, probably by providing melanoma cells with a mechanism to escape from cytotoxic T lymphocyte recognition during disease progression.     

Major histocompatibility complex class I presentation of ovalbumin peptide 257–264 from exogenous sources: protein context influences the degree of TAP-independent presentation

Peritoneal macrophages from C57BL/6 mice process antigens from bacteria or coated on polystyrene beads for presentation by major histocompatibility complex (MHC) class I molecules. To investigate this antigen processing pathway, peritoneal macrophages from homozygous TAP1^−/− mice, which lack the transporter associated with antigen processing (TAP) and are defective in presenting endogenous antigens on MHC class I, were used. TAP1^−/− or C57BL/6 macrophages were co-incubated with either bacteria or polystyrene beads containing the 257–264 epitope from ovalbumin [OVA(257–264)], which binds the mouse class I molecule K^b. The source of the OVA(257–264) epitope was either the Crl-OVA(257–264) (Crl-OVA) fusion protein, the maltose binding protein (MBP)-Crl-OVA fusion protein, native OVA or bacterial recombinant OVA (rOVA); Crl-OVA, MBP-Crl-OVA and rOVA were each expressed in bacteria, and Crl-OVA and MBP-Crl-OVA purified from bacterial lysates and native egg OVA were coated onto polystyrene beads. The data reveal that peritoneal macrophages from C57BL/6 and TAP1^−/− mice can process bacteria expressing Crl-OVA, MBP-Crl-OVA and rOVA as well as beads coated with native OVA, purified Crl-OVA, and purified MBP-Crl-OVA and present OVA(257–264) for recognition by OVA(257–264)/K^b-specific T hybridoma cells, albeit with different relative processing efficiencies. The processing efficiency of TAP1^−/− macrophages co-incubated with bacteria or beads containing Crl-OVA or MBP-Crl-OVA was reduced approximately three to five times compared to C57BL/6 macrophages, but OVA(257–264) was presented 100 times less efficiently when the source of OVA(257–264) was full-length OVA. Chloroquine inhibition studies showed a differential requirement for acidic compartments in C57BL/6 versus TAP1^−/− macrophages, which also depended upon the source of the OVA (257–264) epitope (Crl-OVA versus full-length OVA). These data suggest that TAP1^−/− and C57BL/6 macrophages may process Crl-OVA and full-length OVA in different cellular compartments and that the protein context of the OVA(257–264) epitope influences the extent of TAP-independent processing for MHC class I presentation.     

Linkage disequilibrium between HLA class II (DR, DQ, DP) and antigen processing (LMP, TAP, DM) genes of the major histocompatibility complex

TAP, LMP and DM genes map within the major histocompatibility complex (MHC) class II region between the DQB1 and DPB1 loci, and are involved in the processing of peptides bound to HLA class I or class II molecules. In order to determine the various linkage disequilibria existing between these genes and HLA class II genes, we have analyzed TAP1, TAP2, LMP2, DMA, DMB, DRB1, DQA1, DQB1 and DPB1 polymorphisms in 162 unrelated healthy Caucasian individuals. Many positive or negative associations were observed between alleles at these loci, such as between DR/DQ and TAP2, DM or LMP, between DP and DMB, and between TAP2 and DM, TAP2 and LMP. Conversely, no linkage disequilibrium was detected between some closely related genes (DR/DQ and TAP1, TAP1 and TAP2, LMP2 and DM), in agreement with the existence of recombination hot spots in this region. Other weak linkage disequilibria are likely to exist in this region. These data allow to define some conserved MHC class II haplotypes including HLA class II and TAP, LMP and DM alleles. Furthermore, the knowledge of such linkage disequilibria is of outstanding importance in order to avoid misinterpretation of the data when studying MHC class II associations with autoimmune diseases.