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.     

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.     

Loss of LMP and TAP expression in human melanoma cells

The goal of this study was to determine the frequency of LMP2, LMP7, TAP1 and/or TAP2 loss in melanoma cell lines and in surgically removed melanoma lesions. Cell extracts from control and IFN-γ treated melanoma cell lines were tested in Western blotting with antisera elicited with LMP2, LMP7, TAP1 and TAP2-specific peptides. LMP2 and LMP7 were not detected in 7 out of 9 cell lines. Expression of both LMP subunits was induced in 5 of the 7 negative cell lines by treatment with IFN-γ. TAP1 and TAP2 were not detected in 3 out of 9 cell lines and were induced in one of them by IFN-γ. Surgically removed lesions were stained in the immunoperoxidase reaction with antibodies purified from the antisera by affinity chromatography on the immunizing peptides. LMP2 and LMP7 were not detected in 20% and 6%, respectively, of 32 primary lesions and in 40% and 12%, respectively, of 25 metastatic lesions. TAP1 and TAP2 were not detected in 15% and 18%, respectively, of 32 primary lesions and in 20% and 24%, respectively, of 25 metastatic lesions. Moreover, the frequency of TAP loss is increased in primary lesions from patients with recurrence of their disease, suggesting a potential role in the course of the disease and a negative impact on the outcome of T cell-based immunotherapy.     

TAP1 and TAP2 gene polymorphisms and HLA-TAP haplotypes in Koreans based on 90 families

We have investigated the polymorphism of TAP genes and the distribution of human leukocyte antigen (HLA)-TAP haplotypes in 90 Korean families (180 parents and 233 children), previously typed for HLA class II alleles. TAP1 (codons 333 and 637) and TAP2 (codons 379, 565, 577, 651, 665, and 687) typings were carried out by use of polymerase chain reaction-restriction fragment length polymorphism method. For TAP1, four alleles (gene frequency: A 81.9%, B 15.0%, C 2.5%, D 0.5%) and for TAP2, nine alleles (A1 31.7%, A2 14.2%, B 32.5%, Bky2 6.1%, C 6.9%, D 2.5%, E 3.9%, G 0.8%, and H 1.4%) were detected. Sixteen different TAP1-TAP2 haplotypes were observed with frequencies > 0.5%, and we found that significant linkage disequilibrium was present between TAP1 and TAP2 loci (p < 0.001). When HLA-DRB1-DQB1 haplotypes were extended to TAP1 and TAP2 loci, much diversification of haplotypes was observed: 26 different DRB1-DQB1 haplotypes (frequencies > 0.8%) formed 90 different extended haplotypes. Eight recombinant haplotypes between DQB1 and DPB1 loci were observed, and most (seven of eight) of the recombinations occurred between TAP2 and DPB1 loci. These results add more evidence to the view that recombination hot spot is present within and around TAP gene region.     

Determination of TAP1 and TAP2 polymorphism in the Chinese Han population by real-time TaqMan polymerase chain reaction

The heterodimeric transporter associated with antigen processing (TAP) complex plays a key role in immune surveillance. TAP1 and TAP2 typing was usually performed by polymerase chain reaction (PCR)-restriction fragment length polymorphism and PCR-sequence-specific oligonucleotide probe. As an alternative to these methods, we have established TaqMan assays to determine the frequencies of the TAP1 and TAP2 alleles. We have used these new TaqMan assays to genotype the polymorphisms in 339 unrelated Chinese Hans residing in North and South China. We detected five TAP1 and four TAP2 alleles. All the loci conform to the Hardy-Weinberg expectations. The most frequent alleles in Chinese Hans were TAP1*0101 (79.79%) and TAP2*0101 (82.74%). The two-locus haplotype analysis showed highly significant positive linkage disequilibrium for one TAP1-TAP2 haplotype (TAP1*020101-TAP2*0102), three TAP1-DRB1 haplotypes (TAP1*020101-DRB1*03, TAP1*020102-DRB1*13, and TAP1*0301-DRB1*16), and three TAP2-DRB1 haplotypes (TAP2*0102-DRB1*09, TAP2*0103-DRB1*04, and TAP2*0201-DRB1*01). The three-locus haplotype analysis showed highly significant positive linkage disequilibrium for TAP1*0101-TAP2*0101-DRB1*07, TAP1*0101-TAP2*0103-DRB1*04, TAP1*020101-TAP2*0101-DRB1*03, and TAP1*020101-TAP2*0102-DRB1*13. Comparison of the allele frequencies with those of other populations showed that the TAP1 allele distribution was very similar in all the groups, except for the Guarani, Kaingang, and Anatolian populations, but TAP2 distribution was significantly different from that of the other populations. The new TaqMan method provides relatively accurate, high-resolution, simple, and fast assays for TAP genotyping.     

New Zealand white rabbits immunized with RNA-complexed total histones develop an autoimmune-like response.

The antibody response of rabbits immunized with a total histone mixture containing randomly coiled H1/H5, H2A, H2B, H3 and H4 devoid of DNA was investigated in direct and competitive ELISA. The antisera were tested with isolated histones and chromatin and with a series of overlapping synthetic peptides covering the entire sequences of the four core histones and two peptides of H1. It was found that the New Zealand (NZ) white rabbits immunized with the total histone (TH) mixture complexed with RNA produced IgG antibodies reacting with histones and with a number of histone peptides but not with chromatin. The antisera also contained IgG antibodies which bound components that correspond to common target antigens in autoimmune diseases such as native dsDNA, peptides of Sm-D antigen, ubiquitin, branched peptides of ubiquitinated H2A and poly(ADP-ribose). By competition experiments, it was shown that these antibodies corresponded to non-crossreacting antibody populations. New Zealand rabbits immunized with TH in the absence of RNA or random outbred rabbits immunized with the RNA-complexed histone fraction produced antibodies reacting with histone, chromatin and very few histone peptides, while no activity with non-related antigens was observed. The pattern of reactivity of antisera raised in NZ rabbits with RNA-complexed TH was found to be very similar to that observed in sera of patients with systemic lupus erythematosus while, in contrast, the antibody response was very different in NZ or outbred rabbits immunized with various native nuclear particles and with individual histones. Altered nucleosome particles rather than native nucleosomes may represent the antigenic stimulus giving rise to autoantibodies.     

Expression of HLA class I antigens in transporter associated with antigen processing (TAP)-deficient mutant cell lines

Abstract: Cells lacking expression of the transporter associated with antigen processing (TAP) are deficient in surface HLA class I, yet express reduced levels of HLA-A2 antigen through TAP-independent processing pathways. We have analysed the expression of HLA-A, -B and -C antigens on the 721.174 and T2 TAP-deficient mutant cell lines using a panel of monoclonal antibodies specific for the HLA antigens encoded by the genotype of these cells. Our study has shown the constitutive expression of HLA-Cwl molecules on the cell surface of both T2 and 721.174 cells and has confirmed that HLA-A2 and HLA-B51 are expressed at low levels. Transfection of 721.174 cells with cDNAs encoding TAP1 and TAP2 proteins did not fully restore HLA class I antigen expression on these cells, which appeared to be mainly due to a deficiency in expression of the HLA-B51-associ-ated Bw4 epitope. This suggests that additional antigen-processing genes may be required for optimal generation of HLA-B-binding peptides. Our results indicate that TAP-independent pathways of antigen-processing provide peptides for functional expression of all three classical HLA class I molecules.     

Characterization of rabbit antisera elicited with human LMP2- and LMP7-specific peptides and recombinant proteins

Anti-human LMP2 and anti-human LMP7 sera with a titer of at least 1:10,000 were developed by immunizing rabbits with LMP2- and LMP7-specific peptides corresponding to C -terminal regions of each subunit or with TrxLMP2 and TrxLMP7 recombinant proteins. IgG antibodies elicited by immunization with LMP-specific peptides or recombinant proteins displayed reactivity with their respective immunogens in ELISA. Furthermore, antibodies elicited with both types of immunogens recognize native and recombinant LMP2 and LMP7 subunits in Western blotting and are able to immunoprecipitate LMP2 and LMP7 as components of the 20S proteasome from lymphoid cell lysates. In ELISA, a subpopulation of the antibodies generated with LMP peptides and recombinant proteins corresponding to one LMP subunit is cross-reactive with the other one. This antibody subpopulation was not detectable in the affinity-purified antibody populations isolated by passing antisera over the corresponding immunogen. Neither anti-LMP2 nor anti-LMP7 sera displayed cross-reactivity with the homologous proteasome subunits Delta and MB1. In immunohistochemical reactions affinity-purified anti-LMP2 and anti-LMP7 antibodies stained cells in both frozen and formalin-fixed tissue sections of normal skin. These results indicate that the anti-LMP2 and anti-LMP7 sera elicited with peptides and recombinant proteins are both useful reagents for biochemical characterization of LMP2 and LMP7 and to analyze their expression in normal and transformed cells.     

The human leukocyte antigen TAP2 gene defines the centromeric limit of melanoma susceptibility on chromosome 6p

Abstract: A single human leukocyte antigen (HLA) class II allele, DQB1 * 0301 , is strongly associated with melanoma, and the HLA-DR locus provides the telomeric boundary for melanoma susceptibility in the HLA class II region of chromosome 6. However, the centromeric boundary is unknown. This study was designed to determine whether the adjacent upstream transporter associated with antigen processing (TAP) locus, TAP2 , constitutes the centromeric boundary of disease susceptibility in melanoma. Molecular oligotyping of TAP2 genes was performed for 36 Caucasian patients with melanoma and for 32 Caucasian control individuals by both amplification refractory mutation system (ARMS) polymerase chain reaction (PCR) and PCR-sequence-specific oligonucleotide (SSO) typing. TAP2 allele frequencies in the melanoma patients were compared to those in non-melanoma Caucasian control populations, and to HLA-DQ allele frequencies determined by molecular oligotyping. While HLA-DQB1 * 0301 was more common in this group of 36 melanoma patients compared to a group of 200 controls (56 percent vs. 27 percent, Bonferoni-corrected chi-square p=0.01), no significant differences were observed in TAP2 allele frequencies between melanoma patients and controls. The TAP2 locus represents the centromeric boundary of disease susceptibility for melanoma in the class II region of chromosome 6p. These results support an etiologic role for HLA-DQB1 * 0301 in melanoma susceptibility.     

Major histocompatibility complex class I-restricted alloreactive CD4+ T cells

Although it is well established that CD4+ T cells generally recognize major histocompatibility complex (MHC) class II molecules, MHC class I-reactive CD4+ T cells have occasionally been reported. Here we describe the isolation and characterization of six MHC class I-reactive CD4+ T-cell lines, obtained by co-culture of CD4+ peripheral blood T cells with the MHC class II-negative, transporter associated with antigen processing (TAP)-negative cell line, T2, transfected with human leucocyte antigen (HLA)-B27. Responses were inhibited by the MHC class I-specific monoclonal antibody (mAb), W6/32, demonstrating the direct recognition of MHC class I molecules. In four cases, the restriction element was positively identified as HLA-A2, as responses by these clones were completely inhibited by MA2.1, an HLA-A2-specific mAb. Interestingly, three of the CD4+ T-cell lines only responded to cells expressing HLA-B27, irrespective of their restricting allele, implicating HLA-B27 as a possible source of peptides presented by the stimulatory MHC class I alleles. In addition, these CD4+ MHC class I alloreactive T-cell lines could recognize TAP-deficient cells and therefore may have particular clinical relevance to situations where the expression of TAP molecules is decreased, such as viral infection and transformation of cells.     

Dendritic Cells Engineered to Express Defined Allo-HLA Peptide Complexes Induce Antigen-specific Cytotoxic T Cells Efficiently Killing Tumour Cells

Most tumour-associated antigens (TAA) are non-mutated self-antigens. The peripheral T cell repertoire is devoid of high-avidity TAA-specific cytotoxic T lymphocytes (CTL) due to self-tolerance. As tolerance is major histocompatibility complex-restricted, T cells may be immunized against TAA presented by a non-self human leucocyte antigen (HLA) molecule and transferred to cancer patients expressing that HLA molecule. Obtaining allo-restricted CTL of high-avidity and low cross-reactivity has, however, proven difficult. Here, we show that dendritic cells transfected with mRNA encoding HLA-A*0201, efficiently present externally loaded peptides from the antigen, Melan-A/MART-1 to T cells from HLA-A*0201-negative donors. CD8⁺ T cells binding HLA-A*0201/MART-1 pentamers were detected already after 12 days of co-culture in 11/11 donors. The majority of cells from pentamer⁺ cell lines were CTL and efficiently killed HLA-A*0201⁺ melanoma cells, whilst sparing HLA-A*0201⁺ B-cells. Allo-restricted CTL specific for peptides from the leukaemia-associated antigens CD33 and CD19 were obtained with comparable efficiency. Collectively, the results show that dendritic cells engineered to express defined allo-HLA peptide complexes are highly efficient in generating CTL specifically reacting with tumour-associated antigens.     

HLA class II antigen presentation by melanoma

Constitutive expression of HLA II molecules is normally restricted to professional antigen presenting cells (APCs) of the immune system, although it also occurs in melanoma. Clinical evidence suggests that HLA II expression by melanoma is unfavorable, implicating a role for HLA II expression in the immunopathogenesis of the disease. As a first step, we asked whether human melanoma cells can process antigen or present peptide via HLA II molecules to a peptide specific CD4+ T cell clone. We compared the T cell response to melanoma vs. autologous B cells. In all cell lines tested, melanoma cells were able to process antigen and present peptide efficiently to CD4+ T cells, resulting in T cell proliferation increased 5 to 26 fold over no peptide controls. Blocking CD28 mediated signalling with CTLA-4lg had no effect on the T cell response to either melanoma or B cells. Blocking ICAM-1 resulted in a significant decrease in proliferation (>60%) in response to peptide presentation by melanoma but not B cells. These data demonstrate that (1) HLA II molecules on melanoma cells are functional and antigen processing pathways are intact, and (2) costimulatory signalling mediated via ICAM-1 but not B7 is important in the response of antigen experienced CD4+ T cells to HLA II mediated peptide presentation by melanoma.     

Differential contribution of TAP and tapasin to HLA class I antigen expression

Expression of class I human leucocyte antigens (HLA) on the surface of malignant cells is critical for their recognition and destruction by cytotoxic T lymphocytes. Surface expression requires assembly and folding of HLA class I molecules in the endoplasmic reticulum with the assistance of proteins such as Transporter associated with Antigen Processing (TAP) and tapasin. Interferon-gamma induces both TAP and tapasin so dissection of which protein contributes more to HLA class I expression has not been possible previously. In this study, we take advantage of a human melanoma cell line in which TAP can be induced, but tapasin cannot. Interferon-gamma increases TAP protein levels dramatically but HLA class I expression at the cell surface does not increase substantially, indicating that a large increase in peptide supply is not sufficient to increase HLA class I expression. On the other hand, transfection of either allelic form of tapasin (R240 or T240) enhances HLA-B*5001 and HLA-B*5701 antigen expression considerably with only a modest increase in TAP. Together, these data indicate that in the presence of minimal TAP activity, tapasin can promote substantial HLA class I expression at the cell surface.     

Attempts to optimize active specific immunotherapy for melanoma.

During the past 5 years, we have been conducting clinical trials with a therapeutic melanoma vaccine (melanoma "theraccine"). Mechanical lysates of two melanoma cell lines chosen for their complementary characteristics were combined with the adjuvant DETOX and injected subcutaneously on weeks 1, 2, 3, 4 and 6 for one or two courses, and then monthly in patients with objective clinical responses. Of 109 patients, 22 (20%) have had objective clinical regression of tumor masses, with 5% complete responses. Ten patients have lived more than a year. Eight of the 10 are still alive, five of whom have lived more than 3 years. It was not necessary to achieve complete remissions to cause an increase in survival, and most of the long-surviving patients have one or more (stable) residual nodules. The pace of the disease process has clearly been slowed in those individuals. A rise in the level of cytotoxic T lymphocyte precursors in the blood (pCTL) has correlated with clinical response. Only one patient without such a rise in pCTL has had a response, and assays in that patient were considered unreliable. Both CD4+ and CD8+ CTL have been cloned from the blood of immunized patients. Both types of CTL killed a number of melanoma cell lines, but not other types of tumor or normal cells (lymphoblasts and melanocytes). CD8+ CTL have not been restricted to killing the autologous melanoma. MHC restriction by the HLA-A2 locus was identified. CD4+ CTL were not restricted only by Class II HLA antigens. Many CD4+ clones killed HLA Class II-negative melanomas, and we were able to block cytotoxicity of a particular clone with either anti-HLA Class I or anti-Class II MHC monoclonal antibodies, or both. An association of clinical response to the theraccine with certain HLA phenotypes, notably HLA-C3, -A2 (and the cross-reactive HLA-A28), B12 (and the related alleles (HLA-B44 and -B45) and perhaps DR4, particularly when combinations of those alleles were present, was suggested by our analysis of 70 patients. It is possible that this simply indicates the sharing of MHC antigens between the immunizing melanomas and the patient's melanoma. However, these MHC molecules may be important in their own right in presenting melanoma-associated antigens in CTL in vivo. Subtractive hybridization of mRNA from lung squamous carcinoma cells from cDNA of the M-1 melanoma cell line has yielded several DNA sequences unique to melanoma. Those are now being analyzed for possible immunogenicity, with cytotoxicity by CTL from immunized patients as the major criterion.(ABSTRACT TRUNCATED AT 400 WORDS)     

Analysis of TAP2 polymorphisms in Finnish individuals with type I diabetes

Type I diabetes mellitus is an immune-mediated disease that is known to be associated and linked with genes in the human leukocyte antigen (HLA) region on chromome 6. Functionally, HLA class I antigen presentation may be deranged in type I diabetes. The TAP1 and TAP2 transporters, which mediate the translocation of antigenic peptides into the endoplasmic reticulum and whose genes are located in the HLA class II region, are potential candidates for conferrring predisposition to type I diabetes. Five known coding region variants (codons 379, 565, 651, 665, and 687) as well as three new polymorphisms of TAP2, one silent (codon 604) and two intronic (nucleotide positions 49,270 and 49,471), were typed in a cohort of 146 well-characterized Finnish individuals with type I diabetes and 90 control subjects. Absolute linkage disequilibrium was apparent for the polymorphisms at codons 604, 665, and 687 as well as the two downstream intronic polymorphisms in a 613-bp region of the 3' portion of TAP2; the polymorphism at codon 651, which is also present within this region, was excluded from this linkage. The codon 651 polymorphism defines the allele TAP2F, the frequency of which in HLA-DR4+ diabetic subjects was 5.4 times that in DR4+ controls (27 vs. 5%, p = 0.002, p(c) = 0.01). These data are consistent with the existence of susceptibility haplotypes for type I diabetes in the Finnish population consisting of DRB1*04 (*0401 and *0404), DQ8, and TAP2F.     

Analysis of HLA class I expression in different metastases from two melanoma patients undergoing peptide immunotherapy

We characterized the HLA class I alterations in five metastases obtained from two patients with melanoma immunized with Melan A/MART-1, tyrosinase and gp100 tumor peptides. All three metastases analyzed in the first patient (NW145) showed a similar HLA class I alteration with a dual population of melanoma cells. One population was HLA class I antigen positive and the other had loss of heterozygosity (LOH) in the short arm of chromosome 6 leading to an HLA haplotype loss (A02011, B4007, Cw1). The absence of HLA-A2 antigen may explain why this patient did not develop HLA-A2 restricted, Melan A/MART-1 specificity immunization, since this HLA molecule is the restriction element for the tumor peptides used. However, this HLA-deficient population was not selected after peptide immunotherapy. The primary tumor in this patient presented LOH in region 6q, but only in the vertical growth phase of the lesion, whereas LOH at 6p was observed only in DNA from metastatic material. The second patient (NW16) also presented two metastatic lesions with an identical HLA molecular defect, i.e. HLA B locus downregulation (HLA B51011: serological B51; B1503: serological B70). One lesion expressed the tumor antigen (Melan A/ MART-1), but the other did not. Interestingly, the antigen-positive metastasis regressed after peptide immunotherapy, whereas the other progressed rapidly. These findings provide the first indication that multiple metastases generated in the same host can have identically altered HLA class I phenotypes.     

Immunization with the N-terminal portion of Treponema pallidum repeat protein K attenuates syphilitic lesion development in the rabbit model

When used as an immunogen, Treponema pallidum repeat protein K (TprK) has been shown to attenuate syphilitic lesions upon homologous intradermal challenge in the rabbit model. To further explore this protein as a potential vaccine component, we sought to identify the immunogenic regions of TprK. The abilities of three recombinant peptides encompassing TprK to elicit T- and B-cell responses and to protect against challenge were examined. All three fragments elicited proliferative responses from splenocytes taken from infected rabbits. However, enzyme-linked immunosorbent assays indicated that only fragments 1 and 3 were consistently recognized by antisera from infected rabbits. Each fragment was also used to immunize rabbits that were subsequently challenged intradermally with infectious T. pallidum. All lesions on unimmunized control rabbits ulcerated and contained treponemes, while the lesions on rabbits immunized with fragment 1 were the least likely to have detectable treponemes (25%) and the least likely to ulcerate (37.5%). The lesions on rabbits immunized with fragment 3 showed intermediate results, and rabbits immunized with fragment 2 were the most likely of all those on immunized rabbits to have detectable treponemes (91.7%) and to ulcerate (66.7%). These results demonstrate that epitopes in fragment 1 are recognized by T cells and antibodies during infection and that immunization with this portion of TprK most effectively attenuates syphilitic lesion development.