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PubMedDate not supplied

Comparative assessment of MHC antigen expression in bladder and testis tumour biopsies and established urological tumour cell lines: the relevance of cytokine and gene transfection for correction of defective MHC antigens.

The aim of this study using radio-binding (RB), peroxidase-anti-peroxidase (PAP) and immunoprecipitation (IP) techniques was to investigate the pattern of major histocompatibility complex (MHC) antigen expression in urological malignancies and to compare the results with those seen in established urological human tumour cell lines. The results showed that using PAP technique, the percent bladder cases showing complete loss or cases with greater than 90% of tumour cells negative with W6/32 (detects all class I antigens), HC10 (detects free heavy chain) and BMM.1 (detects beta2-mirogobulin) monoclonal antibodies (Mab) were 16%, 44% and 2% respectively. In a subgroup of 37 cases, the intensity of MHC class II antigen expression for strong, weak and negative cases were 9 (24%), 8 (22%) and 20 (54%) respectively. The expression for class I antigens on testis tumours was mainly negative and when positive, it was present in a small percent of tumour cells. This was also observed for class II antigens where only 8% of cases showed some degree of positivity. Using RB technique, 10 of 12 (85%) of tumour lines expressed class I antigens spontaneously and following interferon gamma (IFNgamma) stimulation, the 2 negative lines one testis (Tera I) and one bladder (Fen) remained negative and 2 lines (both testis lines Tera II and Ep2102) showed a significant class I up-regulation. None of the lines expressed class II antigens spontaneously and following IFNgamma stimulation, 8 of 12 (66%) were induced. The absence of class I and II antigens in the negative lines was confirmed using IP technique. In the case of one class I negative bladder cell line i.e. Fen, the biochemical analysis showed the absence of beta2-m gene product which could not be restored by IFNgamma stimulation. However, transfection of the cells with beta2-m gene resulted in the expression of fully assembled class I antigens, indicating that the loss of antigens was due to the absence of functional beta2-m gene. These results indicated the similarity between the pattern of expression of MHC antigens on tumour biopsies and established tumour cell lines. They also demonstrated that both cytokine stimulation and gene transfection could be used to correct defective class I antigens in tumour cell lines. These approaches might have important implications for pre-selection of bladder cancer patients for cytokine or gene therapies.

Antigens, Neoplasm↗
PubMed2003

High affinity xenoreactive TCR:MHC interaction recruits CD8 in absence of binding to MHC.

The TCR from a xenoreactive murine cytotoxic T lymphocyte clone, AHIII 12.2, recognizes murine H-2D(b) complexed with peptide p1058 (FAPGFFPYL) as well as human HLA-A2.1 complexed with human self-peptide p1049 (ALWGFFPVL). To understand more about T cell biology and cross-reactivity, the ectodomains of the AHIII 12.2 TCR have been produced in E. coli as inclusion bodies and the protein folded to its native conformation. Flow cytometric and surface plasmon resonance analyses indicate that human p1049/A2 has a significantly greater affinity for the murine AHIII 12.2 TCR than does murine p1058/D(b). Yet, T cell binding and cytolytic activity are independent of CD8 when stimulated with human p1049/A2 as demonstrated with anti-CD8 Abs that block CD8 association with MHC. Even in the absence of direct CD8 binding, stimulation of AHIII 12.2 T cells with "CD8-independent" p1049/A2 produces p56(lck) activation and calcium flux. Confocal fluorescence microscopy and fluorescence resonance energy transfer flow cytometry demonstrate CD8 is recruited to the site of TCR:peptide MHC binding. Taken together, these results indicate that there exists another mechanism for recruitment of CD8 during high affinity TCR:peptide MHC engagement.

Animals↗
PubMed1999

Cutting edge: trimolecular interaction of TCR with MHC class II and bacterial superantigen shows a similar affinity to MHC:peptide ligands.

Bacterial superantigens such as Staphylococcus aureus enterotoxin A (SEA) are very potent stimulators of T cells. They bind to the Vbeta region of the TCR and to MHC class II, stimulating T cells at nanomolar concentrations. Using surface plasmon resonance measurements, we find that binding between the individual components of the complex (TCR-class II, TCR-SEA, SEA-class II) is very weak, but that the stability of the trimolecular complex is considerably enhanced, reaching an affinity similar to that found for TCR interactions with MHC:peptide ligand. Thus, the potency of SEA in stimulation of T cells is not due to particularly strong affinities between the proteins, but to a cooperative effect of interactions in the TCR-SEA-MHC class II trimolecular complex that brings the kinetics into a similar range to binding of conventional Ags. This range may be the optimum for T cell activation.

Animals↗
PubMed1990

The predominance of CD8+ T cells after infection with measles virus suggests a role for CD8+ class I MHC-restricted cytotoxic T lymphocytes (CTL) in recovery from measles. Clonal analyses of human CD8+ class I MHC-restricted CTL.

Stimulation of PBMC, in children recovering from acute measles, with autologous EBV-transformed and measles virus (MV)-infected lymphoblastoid cell lines (B-LCL) expanded primarily MV-specific CD8+ T cells. A large number of CD8+ T cell clones were obtained either by passaging of bulk cultures at limiting dilutions or by direct cloning of PBMC without previous stimulation in bulk culture. The MV-specific CD8+ T cell clones responding in a proliferative and a CTL assay were found to be class I MHC restricted. In contrast, CD4+ MV-specific T cell clones, which were generated by the same protocol, recognized MV in association with class II MHC molecules. Analysis of processing requirements for Ag presentation to CD8+ and CD4+ T cell clones, measured by the effect of chloroquine in a proliferative T cell response, revealed that both types of T cells recognized MV Ag processed via the endogenous/cytoplasmic pathway. Thus, these studies indicate that, as in most other viral infections and in contrast to previous suggestions, the class I MHC-restricted CTL response by CD8+ T cells may be an important factor in the control and elimination of MV infection. Therefore, the role proposed for CD4+ class II-restricted T cells in recovery from measles needs to be reevaluated.

Antigen-Presenting Cells↗
PubMed1990

MHC and non-MHC-restricted recognition of filarial surface antigens in mice transplanted with adult Brugia malayi parasites.

We describe here the genetic control of humoral responses to filarial nematode Ag elicited by live adult Brugia malayi parasites in mice. Inbred and congenic mice of two different MHC haplotypes, H-2k and H-2d, were examined. Serologic analysis showed that the humoral responses to the major surface 29-kDa glycoprotein of adult parasites and a 40-kDa Ag from the surface of the microfilarial stage were restricted to mice with H-2k alleles (B10.BR, CBA/Ca, and CBA/N), whereas mice of the H-2d haplotype (B10.D2/n and BALB/c) were nonresponsive to these Ag. Conversely, internal adult Ag of molecular mass of 24 and 66 kDa were recognized only by animals with the H-2d haplotype. Apart from MHC-restricted recognition, the level of responses to phosphorylcholine and to a 15-kDa adult surface molecule were found to be influenced by non-MHC genes. A sharp restriction was also observed to an adult surface Ag complex of 17 to 200 kDa, which was recognized only by BALB/c mice. Thus, multiple examples of both H-2 and background genetic effects on the immune response to distinct filarial Ag can be found.

Animals↗
PubMed1985

GVHR elicited by products of class I or class II loci of the MHC: analysis of the response of mouse T lymphocytes to products of class I and class II loci of the MHC in correlation with GVHR-induced mortality, medullary aplasia, and enteropathy.

A lethal graft-vs-host reaction (GVHR) was elicited by the injection into irradiated (700 rad) mice, reconstituted with T-depleted bone marrow cells (BM), of T lymphocytes incompatible for different loci of the major histocompatibility complex (MHC). The number of T cells needed to kill more than 50% of the recipients by day 40 was about 10(6) for GVHR elicited across the product of the K, D, or E locus, but about 10(5)--10--fold less-when the A locus was involved. The mortality was associated with a medullary aplasia in all strain combinations, but enteropathy was observed only in GVHR elicited by the products of class II, and not class I, loci. Mortality and medullary aplasia were diminished or absent in recipients reconstituted with BM cells from T cell donors instead of cells of the host genotype, which suggests a direct (cytolytic) T-hematopoietic cell interaction. Lymphoproliferation was evident within the host spleen and lymph node 5 days after injection of T lymphocytes incompatible for class II but not class I loci. Spleens from mice suffering from a lethal GVHR were examined by culture in limiting dilution to evaluate the frequency of anti-host T cells and to derive anti-host T cell clones and lines, whose properties were explored. In the GVHR elicited across the A or E region of the MHC, examined between days 7 and 19, a high frequency (10(-2] of anti-host cells was observed. The polyclonal cell lines isolated (16) all displayed MLR responsiveness, antigen-driven IL 2 production, and cytolysis for LPS blasts of the host genotype. However, among 13 clones isolated, two categories were observed: Lyt-2-, which were MLR responders and IL 2 producers (four of 13), and Lyt-2+, which were cytolytic but neither MLR responders nor IL 2 producers (nine of 13). In the GVHR elicited by the K or D region, examined between days 7 and 90, the frequency of anti-host cells was low (10(3) to 10(4], with a tendency to decrease during the progression of the disease. The lines (11) or clones (26) isolated from different mice were all Lyt-2+ and strongly cytolytic but proliferated poorly and produced no IL 2 in MLR. These findings suggest that the Lyt-2+ lymphocytes, recognizing the products of the class I loci, function in vivo without proliferation and without requiring helper T cells. Cell lines specific for class I or class II loci of the MHC produced interferon and colony-stimulating factors.

Animals↗
PubMed1994

MHC class I and non-MHC-linked capacity for generating an anti-viral CTL response determines susceptibility to CTL exhaustion and establishment of virus persistence in mice.

The influence of the murine MHC gene complex H-2 class I alleles or of the genetic background and the role of virus variants on establishment of a persistent infection with lymphocytic choriomeningitis virus (LCMV) was analyzed in immunocompetent mice of H-2d haplotype by evaluation of cytotoxic T cell responses. Susceptibility to establishment of a virus carrier state increased in various H-2d mice with lower CTL response and slower CTL kinetics. Low responder BALB/c-H-2dm2, lacking H-2Ld molecules to present the major T cell epitope amino acids 118-126 of the LCMV nucleoprotein or DBA/2 mice possessing relatively few CD8+ T cells, were more susceptible than high responder BALB/c mice expressing H-2Ld. Additional critical factors were LCMV isolate and dose of infection. The rapidly replicating LCMV-DOCILE and Cl 13 Armstrong induced viral persistence readily, whereas the slowly replicating parental virus strains WE or Armstrong did not. The presented findings illustrate a model of MHC-linked or MHC-unlinked susceptibility for virus persistence and may help to explain pathogeneses of chronic virus infections in humans that are often associated with slowly progressing immunopathology.

Animals↗
PubMed1996

The role of MHC- and non-MHC-associated genes in determining the human immune response to malaria antigens.

Individual susceptibility to malaria infection, disease and death is influenced by host genotype, parasite virulence and a number of environmental factors including malaria-specific immunity. Immune responses are themselves determined by a combination of host genes and environmental effects. The extent to which host genotype limits the spectrum of possible immune responses may influence the outcome of infection and has consequences for vaccine design. Associations have been observed between human major histocompatibility complex (MHC) genotype and susceptibility to severe malaria, but no similar associations have been observed for mild malarial disease or for specific antibody responses to defined malaria antigens. Epidemiological studies have shown that, in practice, neither T helper cell nor antibody responses to malaria parasite are limited by host MHC genotype, but have revealed that genes lying outside the MHC may influence T cell proliferative responses. These genes have yet to be identified, but possible candidates include T cell receptor (TcR) genes, and genes involved in TcR gene rearrangements. More importantly, perhaps, longitudinal epidemiological studies have shown that the anti-malarial antibody repertoire is selective and becomes fixed in malaria-immune individuals, but is independent of host genotype. These findings suggest that the antibody repertoire may be determined, at least in part, by stochastic events. The first of these is the generation of the T and B cell repertoire, which results from random gene recombinations and somatic mutation and is thus partially independent of germline genes. Secondly, of the profusion of immunogenic peptides which are processed and presented by antigen presenting cells, a few will, by chance, interact with T and B cell surface antigen receptors of particularly high affinity. These T and B cell clones will be selected, will expand and may come to dominate the immune response, preventing the recognition of variant epitopes presented by subsequent infections-a process known as original antigenic sin or clonal imprinting. The immune response of an individual thus reflects the balance between genetic and stochastic effects. This may have important consequences for subunit vaccine development.

Animals↗
PubMedDate not supplied

Major histocompatibility complex (MHC)-I and MHC-II expression in Hodgkin's disease in relation to the presence of Epstein-Barr Virus (EBV).

We have investigated the immunohistochemical expression of beta2-microglobulin and HLA-DR proteins in Hodgkin's disease (HD) in relation to the expression of the EBV-encoded EBER1-2 mRNAs and the LMP-1 protein. beta2-microglobulin is expressed in association with MHC-I molecules on most nucleated cells and HLA-DR belongs to the MHC-II molecules which are expressed mostly on antigen-presenting cells. Formalin-fixed paraffin embedded tissue from 39 cases of lymphonodal HD were stained by immunohistochemistry for beta2-microglobulin, HLA-DR and LMP-1 proteins and by RNA in situ hybridization for EBER1-2 mRNAs. beta2-microglobulin positive staining was found in Reed-Sternberg and Hodgkin cells (HRS cells) in 18/39 cases of HD. HLA-DR positive staining was found in HRS cells in all cases of HD. EBER1-2 transcripts and LMP-1 protein were detected in HRS cells in 16/39 cases of HD. No correlation as found between the presence of EBER 1-2 transcripts or the LMP-1 protein and the detection of beta2-microglobulin and HLA-DR proteins in HD. Thus, EBV does not seem to use downregulation of MHC-I to avoid the T-cell cytotoxic immune response in HD. In addition, EBV does not seem to be the only factor responsible for the HLA-DR expression in HRS cells of HD, although it could participate in the induction of the expression of HLA-DR molecule in the EBV positive cases of HD.

Herpesvirus 4, Human↗
PubMed1984

MHC homology between various mammalian species at the DNA level: its relevance to MHC evolution.

Considerable homology concerning the major histocompatibility complexes of man, rhesus monkey, dog, cat, cow, sheep, rabbit and goat has been established by Southern blot analyses. Human cDNAs from both class I and class II gene products were used as probes. Besides the expected hybridization of the probes to human DNA, the probes displayed a clear affinity for the other DNAs as well. Equal numbers of bands-about 16 depending on the enzyme used - appear in digests of human and rhesus monkey DNA when the class I cDNA is used as a probe. This number is slightly smaller and varying in the other species. The same holds for class II probes. Both class I and class II probes show no detectable homology at all with Xenopus DNA under the conditions used. A substantial MHC homology at the DNA level between man and rhesus monkey can be concluded from these data, this homology being somewhat smaller in various other species. Further implications for the evolution of MHC are discussed.

Animals↗
PubMed1997

Molecular aberrations in the MHC class I-restricted pathway for antigen presentation in methylcholanthrene sarcomas from nude mice: discrepancies between MHC mRNA and surface protein.

In a previous study, we demonstrated that eight sarcomas induced by chemical carcinogenesis in nude mice were rejected by syngeneic immunocompetent recipients at a much higher rate than eight sarcomas induced with the same method in syngeneic immmunocompetent mice. In the present study, we investigated these 16 sarcomas for structural and quantitative aberrations in components of the MHC class I-restricted antigen-processing and -presentation pathway. Considerable discrepancies between mRNA levels and cell surface protein expression of MHC class I (Kd, Dd and Ld) molecules were observed almost exclusively in the tumours derived from nude mice. Several of the nude mouse-derived tumours also displayed incongruent levels of heavy chain mRNA and beta2-microglobulin mRNA. These findings are taken as indications of abnormal regulation of gene transcription in nude mouse tumours, and if this abnormal regulation extends to the entire genome, it may explain the pronounced immunogenicity of these tumours. Proteasome composition, heat shock protein expression, TAP-molecule inducibility and intercellular adhesion molecule-1 expression were investigated in the same tumours. We found no indications of structural defects or quantitative differences in these molecules between the two groups of tumours.

Animals↗
PubMed1985

Genetic susceptibility to murine collagen II autoimmune arthritis. Proposed relationship to the IgG2 autoantibody subclass response, complement C5, major histocompatibility complex (MHC) and non-MHC loci.

Analysis of the IgG autoantibody subclass response in the collagen II autoimmune arthritis (CII AIA)-susceptible D1 strain mice revealed that the onset of disease was associated with a predominance of IgG2a autoantibody. In a comparative study, resistance in the B6 strain was associated with a deficient IgG2a autoantibody response. B6 IgG1, 2b, and 3 autoantibody responses generally overlapped those of arthritic D1 mice, and estimates of antibody crossreactivity and affinity were similar for both strains. In crosses between D1 and B6, arthritis developed only in those F1 mice with IgG2a autoantibody responses approximating or exceeding those in arthritic D1 mice. Additional studies with B6 and B10 strains suggested an alternate role for the IgG2b autoantibody response. In inbred strains with known genetic backgrounds, a dissociation between the magnitude of the total IgG autoantibody response and the percent of total as IgG2a was demonstrated. The H-2q, Ig-1c D1 strain was a high-total and high-percent IgG2a responder, while the H-2d, Ig-1c D2 strain was a low-total but high-percent IgG2a responder. The H-2b, Ig-1b B6 strain was a low-total and low-percent IgG2a responder, while the H-2b/q, Ig-1b/c (B6D1)F1 hybrid was a low-total but high-percent IgG2a responder. A further dissociation between high-percent IgG2a autoantibody responsiveness and the H-2 haplotype was demonstrated by the H-2 congenic B10.D2/n (H-2d, Ig-1b) strain, in which a low-percent IgG2a response was observed to differ from the D2 strain. High-percent IgG2a autoantibody responsiveness also appeared to be inherited as a dominant trait based upon high responses in all (B6D1)F1 hybrids and backcrosses to D1. These findings suggest that the H-2 haplotype is involved in the total IgG autoantibody response but that the relative fraction of the total response as IgG2a is independent of the H-2 haplotype and possibly related to Igh-C genes. C5-deficient SWR (H-2q, Ig-1c) mice were found to have a high total autoantibody response to mouse CII and IgG2a comparable to arthritic D1 mice, but these mice did not develop arthritis. Based upon these observations, we conclude that susceptibility to CII AIA requires the interaction of multiple genes, both major histocompatibility complex (MHC) and non-MHC, which influence the magnitude (total IgG) and the quality (IgG subclass) of the autoimmune response and the availability of appropriate mediators (C5) to initiate the inflammatory reaction.

Animals↗
PubMed1992

Tolerance and MHC restriction in transgenic mice expressing a MHC class I gene in erythroid cells.

Transgenic mice carrying a MHC class I structural gene (H-2Kb) linked to transcriptional control elements from the human beta-globin gene, which direct erythroid lineage specific transcription, express H-2Kb molecules in red blood cells but H-2Kb expression cannot be detected in skin or lymphoid cells. This limited pattern of self MHC expression is sufficient to induce tolerance to H-2Kb molecules and H-2Kb restricted cytotoxic T cell responses can be generated in transgenic mice. Transgenic mice are unable to mount H-2Kb specific cytotoxic T cell responses in vitro, even when exogenous IL-2 is provided. However, H-2Kb specific T cell proliferative responses are comparable with H-2Kb specific responses in non-transgenic mice, even in the absence of exogenous IL-2. Thus, expression of H-2Kb molecules under control of human beta-globin transcriptional control elements in transgenic mice is tolerogenic but does not result in elimination of all H-2Kb reactive T cells from the mature repertoire. This suggests that tolerance in these mice may arise due to functional inactivation of H-2Kb reactive T cells in vivo when they encounter H-2Kb molecules expressed on cells of erythroid cell lineages or on non-erythroid cells which express H-2Kb molecules at very low levels or in a developmentally regulated pattern. Furthermore, in spite of the failure to detect H-2Kb expression on non-erythroid cells in these mice, we conclude that H-2Kb molecules participate in positive selection of the T cell repertoire since H-2Kb restricted T cell responses can be generated in these transgenic mice.

Animals↗
PubMed1981

Complementing MHC- and non-MHC-linked genes and resistance to avian sarcoma virus-induced tumours in inbred lines of chickens.

Schmidt-Ruppin Rous sarcoma virus, subgroup B (SR-RSV-B), was inoculated into the wingwebs of chickens from three partially congenic inbred lines, G-B1, G-B2 and G-B3, homozygous for different MHC (B region) haplotypes (genotypes = B1/B1, B2/B2 and B3/B3 respectively). All birds developed tumours but only the G-B2 line resisted progressive tumour growth. Birds from lines G-B1 and G-B3 approached 100% susceptibility to progressive tumour growth, whereas most (G-B1 X G-B3) F1 hybrids were resistant to tumours induced by SR-RSV-B. The association of the resistance trait in F1 hybrids with genes of the B region was investigated by testing progeny of (G-B1 X G-B3) X B-B1 F1 and (G-B1 X G-B2) X G-B3 F1 backcross matings. Approximately 27% of the backcross population was resistant to SR-RSV-B-induced tumours and these resistant offspring were predominantly of the B1/B3 phenotype. We interpret these results to mean that resistance to progressive tumour growth involves complementation between genes (allelic or at separate loci) linked to or within the B region and that resistance is effective only when the complementing B region genes act in concert with complementing genes which assort independently of the MHC. We suggest that complementing B region-linked genes are homologues of complementing murine H-2-linked Ir genes. The function of the B region in determining growth of sarcomas may therefore be analogous to that of Ir genes.

Alleles↗
PubMed2001

Epitope mapping of the indirect T cell response to allogeneic class I MHC: sequences shared by donor and recipient MHC may prime T cells that provide help for alloantibody production.

Indirect allorecognition occurs when T cells recognize donor MHC presented as peptide epitopes by recipient APC, but the precise nature of the epitopes involved remains unclear. Rejection of rat MHC class I-disparate PVG.R8 (RT1.A(a)) grafts by PVG.RT1(u) (RT1.A(u)) recipients is mediated by indirectly restricted CD4 T cells that provide help for the generation of alloantibody. In this study, epitope mapping was performed using a functionally relevant readout (alloantibody production) to identify key peptides that prime an indirect alloimmune response, leading to graft rejection. PVG.RT1(u) rats were immunized with a series of overlapping 15-mer peptides (peptides 1-18) that spanned the alpha1 and alpha2 domains of the RT1.A(a) molecule. Several peptides were able to accelerate both the alloantibody response to the intact RT1.A(a) Ag and PVG.R8 heart graft rejection. An immunodominant epitope was identified within the hypervariable region of the alpha1 domain. Fine mapping of this region with a second series of peptides overlapping by single amino acids confirmed the presence of an eight-amino acid core determinant. Additional "subdominant" epitopes were identified, two of which were located within regions of amino acid homology between the RT1.A(a) and RT1.A(u) molecules and not, as had been expected, within other hypervariable regions. The contribution of self-epitopes to indirect allorecognition was emphasized by the demonstration that i.v. administration of a 15-mer peptide encompassing one of the subdominant self-determinants diminished the recipient's ability to mount an alloantibody response on challenge with intact A(a) alloantigen. Our findings suggest that cryptic self-epitopes recognized by autoreactive T cells may contribute to allograft rejection and should be considered when designing novel strategies for inducing tolerance to alloantigen.

Amino Acid Sequence↗
PubMed1995

Structure of the MHC A and B locus promoters in hominoids. Insights on the evolution of the class I MHC multigene family.

The expansion and contraction of mammalian class I multigene families raises the issue as to what determines the loss or retention of family members. We propose that accumulating changes in regulatory regions result in the loss of expression of the gene products during times critical to selection, leading to the extinction of misregulated genes. The structures of promoter regions of MHC class I genes in nonhuman primates support this view. The B promoters are more homogeneous and contain regulatory elements also found in the promoters of the homologous class I genes of more distant mammals, whereas the A locus promoters were significantly more heterogeneous, have fewer sequence motifs related to known transcription factor-binding sites and have accumulated nucleotide substitutions within one of the widely conserved class I promoter elements. These findings are consistent with the view that the more polymorphic B locus is the principal MHC locus encoding functional class I Ag-presenting molecules whereas the less polymorphic A locus is assuming a secondary role as a consequence of promoter defects.

Animals↗
PubMed1994

Gene required for normal MHC class II expression and function is localized to approximately 45 kb of DNA in the class II region of the MHC.

In certain mutant human B cell lines, MHC-encoded class II molecules displayed at the cell surface have an abnormal conformation and are unstable in the presence of SDS. The mutants cannot present exogenous protein Ags to T cells but elicit responses with exogenous antigenic peptides; thus, formation of intracellular complexes between antigenic peptides and class II molecules is impaired. Previous analysis of LCL deletion mutants, .82, .174, and 5.2.4, showed that genes needed for this function must be present in approximately 230 kb of DNA in the class II region of the MHC. We now describe a new deletion mutant, .61, which has normal class II-mediated Ag processing/presentation. The TAP1, TAP2, LMP2, and LMP7 genes are deleted from .61, demonstrating that those genes are not needed for normal formation of intracellular class II/peptide complexes. The genes in question must be located in DNA that is present in .61 and .82 (both normal) and absent from .174 and 5.2.4. (both defective). Mapping of the deletion breakpoints indicates that genes needed for normal class II-associated Ag processing/presentation are either: 1) in an approximately 40 kb L DNA segment located between the DMB and LMP2 loci or 2) in an R region between the DQA2 and DQB1 loci and are completely included on a 5.1-kb fragment formed by joining of DNA that flanks the deletion in .61. The evidence favors location of the genes in the L DNA segment.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed2000

Recombinant human single-chain MHC-peptide complexes made from E. coli By in vitro refolding: functional single-chain MHC-peptide complexes and tetramers with tumor associated antigens.

Soluble recombinant MHC-peptide complexes are valuable tools for molecular characterization of immune responses as well as for other functional and structural studies. In this study, soluble recombinant single-chain human MHC (scMHC)-peptide complexes were generated by in vitro refolding of inclusion bodies from bacterially expressed engineered HLA-A2 in the presence of tumor-associated or viral peptides. The scMHC molecule was composed of beta2-microglobulin connected to the first three domains of the HLA-A2 heavy chain through a 15-amino acid flexible linker. Highly purified scMHC-peptide complexes were obtained in high yield using several peptides derived from the melanoma antigens gp100 and MART-1 or a viral peptide derived from HTLV-1. The scMHC complexes were characterized in detail and were found to be correctly folded and able to specifically bind HLA-A2-restricted peptides. We also generated scMHC-peptide tetramers, which were biologically functional; they induced a peptide-specific CTL clone to be activated and secrete IFN-gamma, and were able to stain specifically CTL lines. Such recombinant soluble scMHC-peptide complexes and tetramers should prove of great value for characterization of immune responses involving CTL, for visualization of antigen-specific immune responses, for in vitro primary CTL induction, and for peptide binding assays and structural studies.

Antigens, Neoplasm↗