Biomedical subjects

J McCluskey

Publications and source records attributed to J McCluskey.

At least 37 records · Page 2Linked to original sources

PubMed1999

Matching for HLA DPA1 and DPB1 alleles in unrelated bone marrow transplantation.

The impact of donor-recipient DPA1 and DPB1 matching was examined in 122 unrelated bone marrow transplant pairs. All pairs were serologically matched at the time of transplantation for HLA class I and II and a majority also DRB1 allele matched. Retrospective A, B, C, DRB1, DQA1, DQB1 in addition to DPA1 and DPB1 allele matching was performed by molecular techniques. The percentage of pairs that were allele matched was as follows; HLA-A = 91% (n = 80), HLA-B = 94% (n = 80), HLA-C = 78% (n = 80), HLA-DRB1 = 96% (n = 122), HLA-DQA1 = 99% (n = 80), HLA-DQB1 = 92% (n = 122). 92 recipient/donor pairs with informative clinical data were available for analysis. DPA1 identity (no incompatibility in either direction) was observed in 57% and DPA1 compatibility in 76% of pairs with no apparent beneficial effect of matching on patient survival or Graft Versus Host Disease (GVHD). DPB1 identity was observed in 11% and compatibility in 27% of pairs. A significant improvement in patient survival was observed in DPB1 matched compared to one DPB1 mismatch (p < 0.01) and combined one and two DPB1 mismatched transplants (p = 0.03). This beneficial effect remained when allele mismatches at HLA-A, B, C, DRB1, DQA1, DQB1 were excluded (p = 0.05, p = 0.03, respectively). There was a significant association of increased frequency of severe GVHD (grades III-IV) compared to mild GVHD (grades I-II) with DPB1 mismatched transplants compared to DPB1 matched transplants (p = 0.04). In DPB1 mismatched transplants an association between patient survival and matching for individual DPB1 polymorphic regions was not observed; however in the HLA-A, B, DRB1, DQA1, DQB1 allele matched transplants a non significant increase in the frequency of Grade IV GVHD was observed in recipients who were negative compared to those who were positive for DPB1 alleles coding for glutamic acid at position 69.

Adult↗

PubMed1999

HFE genotypes and haemochromatosis: quantifying the risks of disease.

Hereditary haemochromatosis (HH) is an autosomal recessive disease involving mutations in the recently characterised HFE gene linked to HLA-A in the major histocompatibility complex. The known HFE polymorphisms include the wild-type allele, a G-->A substitution at base 845 (845A) and a C-->G substitution at position 187 (187G). Although most cases of HH are accountable by homozygosity of the 845A allele the exact risk of other HFE genotypes, especially those involving the 187G allele has not been determined. We have compiled estimates of disease risk for all known HFE genotypes by re-analyzing published studies. The data show a hierarchical risk calculated as odds ratio (OR) for each genotype 845A/ 845A (OR=2101); 845A/187G (OR=24); 187G/187G (OR=9); 845A/Wt (OR=5); 187G/Wt (OR=2). Interestingly, the disease risk of 187G-genotypes suggests that subtle functional changes in the HFE product can interact with other genetic factors (e.g. trans allele, gender) and environmental factors (e.g. diet) to manifest either as clinical disease, altered iron stores or a normal phenotype. This paradigm is potentially useful in understanding the contribution of HLA alleles to risk of various disorders especially autoimmunity.

Genetic Linkage↗

PubMed1999

Molecular chaperones are targets of autoimmunity in Ro(SS-A) immune mice.

We have used a murine model of experimental anti-Ro(SS-A) autoimmunity to dissect additional intermolecular interactions between the 52-kD Ro (Ro52) and 60-kD Ro (Ro60) autoantigens and molecular chaperones. Immune responses to members of the heat shock protein hsp70 and hsp90 families were measured by immunoblotting and ELISA in sera from mice immunized and boosted with purified recombinant Ro52, Ro60 and La (SS-B). All Ro52 and Ro60 immune sera immunoblotted the inducible glucose-regulated protein grp78 and hsp70 species but not constitutive hsc70 or hsp90. The kinetics of antibody production and reciprocal affinity purification experiments indicated that the grp78 and hsp70 responses were cross-reactive but distinct from immune responses to the primary Ro52 and Ro60 immunogens and the endoplasmic reticulum (ER)-resident chaperone calreticulin. No responses to molecular chaperones were detected in the La-immunized mice. Control immunizations indicated that the recruited grp78 and hsp70 responses were specific for the Ro proteins and not due to immunization with denatured protein. The rapid spreading of immunity to the inducible grp78 and hsp70 in Ro52- and Ro60-immunized mice suggests that these components may co-localize and physically associate under certain physiological conditions which may promote autoimmunization. The potential importance of the ER-resident chaperones grp78 and calreticulin is further supported by their co-localization with Ro in small apoptotic membrane blebs and the finding of a novel putative grp78 binding motif in the carboxyl-terminal region of Ro52.

Animals↗

PubMed1999

The human leucocyte antigens and clinical medicine: an overview.

The Major Histocompatibility Complex (MHC) occupies 4-6 megabases on the short arm of chromosome 6 and is the most intensively studied segment of the human genome. This region was first discovered through its influence on transplantation rejection and on antigen-specific immune responses. The most important genes for managing these functions encode the HLA molecules (human leucocyte antigens) which are highly polymorphic in human populations. HLA typing for these polymorphisms is widely used in clinical medicine when identifying optimal organ donors or recipients and in assessing the risk of diseases such as narcolepsy, hereditary hemochromatosis, ankylosing spondylitis and certain autoimmune disorders. As new genes are identified in the MHC, the clinical impact of this genetic region is likely to assume further importance as outlined in this review.

Antigen Presentation↗

PubMed1998

Avoidance of self-reactivity results in skewed CTL responses to rare components of synthetic immunogens.

In studying the CTL recognition of peptide determinants derived from the nuclear Ag La (SS-B), we observed significant skewing of the response toward rare components present within the immunogen. Thus, priming of naive mouse lymphocytes in vitro with a synthetic H-2Kb-binding peptide comprising human La (hLa) residues 51-58 resulted in class I-restricted cytotoxic T cells that failed to recognize naturally presented hLa 51-58 peptide. Instead, the majority of T hybrids recognized a low abundance (< or = 1%) contaminant present at picomolar concentrations in the original synthesis and identified as a peptide adduct containing N,4-t-butyl asparagine at position 6 of the hLa 51-58 sequence. The preferred T cell recognition of the butyl adduct was not due to increased affinity of this peptide for the H-2Kb molecule or to the antagonism of CTL recognizing the unmodified determinant. Rather, the bias in the immune response appeared to be the result of partial self-tolerance to the homologous mouse La 51-58 determinant, which differs from its human counterpart by only a single amino acid at position 1 (T-->I). Accordingly, the CTL response appeared to be focused on "non-self" ligands present within the synthesis, even though they were present at very low concentrations. These observations have significant implications for the use of synthetic peptide vaccines, especially those designed to manipulate responses to self peptides such as tumor Ags in which self-tolerance may result in unexpected reactivity.

Amino Acid Substitution↗

PubMed1998

Control of cell cycle gene expression in bone development and during c-Fos-induced osteosarcoma formation.

We have used c-Fos transgenic mice which develop osteosarcomas to determine the expression patterns of cyclins, cyclin-dependent kinases (CDKs), and cyclin-dependent kinase inhibitors (CKIs) in different bone cell populations in order to define the potential mechanisms of c-Fos transformation. Immunohistochemical analysis in embryonic and early postnatal bone demonstrated that cyclin E and its kinase partner CDK2 were expressed specifically in bone-forming osteoblasts. Cyclin D1 expression was absent despite high levels of CDK4 and CDK6, and the CKI p27 was expressed in chondrocytes, osteoclasts, and at lower levels in osteoblasts. Following activation of the c-fos transgene in vivo and before overt tumor formation, cyclin D1 expression increased dramatically and was colocalized with exogenous c-Fos protein specifically in osteoblasts and chondrocytes, but not in osteoclasts. Prolonged activation of c-Fos resulted in osteosarcoma formation wherein the levels of cyclin D1, cyclin E, and CDKs 2, 4, and 6 were high in a wide spectrum of malignant cell types, especially in transformed osteoblasts. The CKI p27 was expressed at very high levels in bone-resorbing osteoclasts, and to a lesser extent in chondrocytes and osteoblasts. These in vivo observations suggest that cyclin D1 may be a target for c-Fos action and that elevation of cyclin D1 in osteoblasts which already express cyclin E/CDK2 and the cyclin D1 partners CDKs-4 and 6, may predispose cells to uncontrolled cell growth leading to osteosarcoma development. This study implicates altered cell cycle control as a potential mechanism through which c-Fos causes osteoblast transformation and bone tumor formation.

Animals↗

PubMed1998

Molecular basis of IgE-recognition of Lol p 5, a major allergen of rye-grass pollen.

Grass pollen, especially of rye-grass (Lolium perenne). represents an important cause of type I allergy. Identification of IgE-binding (allergenic) epitopes of major grass pollen allergens is essential for understanding the molecular basis of interaction between allergens and human IgE antibodies and therefore facilitates the devising of safer and more effective diagnostic and immunotherapy reagents. The aim of this study was to identify the allergenic epitopes of Lol p 5, a major allergen of rye-grass pollen, immunodissect these epitopes further so that the amino acid residues critical for antibody binding can be determined and investigate the conservation and nature of these epitopes within the context of the natural grass pollen allergens. Peptides, 12-13 amino acid residues long and overlapping each other by 4 amino acid residues, based on the entire deduced amino acid sequence of the coding region of Lol p 5, were synthesised and assayed for IgE-binding. Two strong IgE-binding epitopes (Lol p 5 (49-60) and (265-276), referred to as peptides 7 and 34, respectively) were identified. These epitopes were further resolved by truncated peptides and amino acid replacement studies and the amino acid residues critical for IgE-binding determined (Lol p 5 (49-60) residue Lys57 and (265-276) residue Lys275). Sequences of these epitopes were conserved in related allergens and may form the conserved allergenic domains responsible for the cross-reactivity observed between pollen allergens of taxonomically related grasses. Furthermore, due to its strong IgE-reactivity, synthetic peptide Lol p 5 (265-276) was used to affinity-purify specific IgE antibodies which recognised proteins of other clinically important grass pollens. further indicating presence of allergenic cross-reactivity at the level of allergenic epitope. Moreover, Lol p 5 (265 276) demonstrated a strong capacity to inhibit IgE-binding to natural rye-grass pollen proteins highlighting the antibody accessibility to these sequences within the context of the natural allergens. Strong IgE-binding epitopes of Lol p 5 have been identified down to single critical amino acid residues and are shown to occur as linear or continuous domains in the natural conformation of natural Lol p 5 and other group 5 grass pollen allergens. The fact that such an allergenic synthetic epitope has the capacity to strongly inhibit IgE-binding to natural allergens highlight its potential for use as a candidate in future therapeutics to treat pollen-associated allergies.

Allergens↗

PubMed1998

HLA-B27-restricted antigen presentation in the absence of tapasin reveals polymorphism in mechanisms of HLA class I peptide loading.

Tapasin is a resident ER protein believed to be critical for antigen presentation by HLA class I molecules. We demonstrate that allelic variation in MHC class I molecules influences their dependence on tapasin for peptide loading and antigen presentation. HLA-B*2705 molecules achieve high levels of surface expression and present specific viral peptides in the absence of tapasin. In contrast, HLA-B*4402 molecules are highly dependent upon human tapasin for these functions, while HLA-B8 molecules are intermediate in this regard. Significantly, HLA-B*2705 like HLA-B*4402, requires tapasin to associate efficiently with TAP (transporters associated with antigen processing). The unusual ability of HLA-B*2705 to form peptide complexes without associating with TAP or tapasin confers flexibility in the repertoire of peptides presented by this molecule. We speculate that these properties might contribute to the role of HLA-B27 in conferring susceptibility to inflammatory spondyloarthropathies.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1998

HLA class II phenotype controls diversification of the autoantibody response in primary Sjögren's syndrome (pSS).

The coexistence of anti-La (SS-B) and anti-Ro (SS-A) autoantibodies in pSS is probably explained by intermolecular spreading of autoimmunity toward different components of the La/Ro ribonucleoprotein (RNP). In order to evaluate the role of the HLA class II phenotype in controlling diversification of this autoantibody response, 80 patients with pSS were typed by polymerase chain reaction sequence-specific oligonucleotide (PCR-SSO) at the HLA class II loci DRB1, DQA1 and DQB1. Serum samples were examined for anti-La and anti-Ro by counterimmunoelectrophoresis and by ELISA using purified recombinant La and 60-kD Ro proteins. Patient sera were classified according to the extent of diversification of the anti-La, anti-Ro response including the presence or absence of precipitating anti-La antibodies. Immunogenic characteristics of these stratified groups were then studied. All patients with pSS, with or without autoantibodies to Ro and La, were found to have at least one of the HLA-DRB1 types DR2, DR3 or DR5. The HLA DR3-DQA1*0501-DQB1*02 (DR3-DQ2) haplotype was primarily associated with a diversified La/Ro RNP response containing precipitating autoantibodies to La (P<0.001); whereas the haplotype HLA DR2-DQA1*0102-DQB1*0602 (DR2-DQ1) was associated with a less diversified La/Ro RNP response containing non-precipitating (restricted epitope) anti-La autoantibodies (P<0.001). Anti-La-positive patients lacking both HLA-DR2 and HLA-DR3 all expressed the HLA-DQA1*0501 allele, which was present at increasing frequency with greater diversification of the anti-La/Ro autoantibody response. The association of distinct HLA haplotypes with different degrees of autoantibody diversification in patients with pSS suggests a model of HLA-restricted presentation of La/Ro peptide determinants to autoreactive helper T cells. We propose that non-precipitating anti-La responses are driven by limited intermolecular help from DR2-DQ1-restricted T helper cells recognizing Ro determinants. On the other hand, we speculate that the more diversified, precipitating anti-La responses obtain more efficient cognate T help from DR3-DQ2-restricted T helper cells recognizing La determinants, where HLA-DQA1*0501 may be a critical determinant for antigen presentation.

Alleles↗

PubMed1998

Determinant spreading: lessons from animal models and human disease.

Spreading of the immune response is a common theme in organ-specific and systemic autoimmune diseases. We evaluated whether some of the mixed antinuclear antibody patterns characteristic of systemic autoimmunity might be the result of determinant spreading from a single initiating event. Immunisation of healthy mice with individual protein components of the La/Ro ribonucleoprotein (RNP) targeted in systemic lupus erythematosus and primary Sjögren's syndrome induced autoantibodies recognising Ro60 (SS-A), Ro52 (SS-A) and La (SS-B) and in some cases the molecular chaperones calreticulin and Grp78. The endogenous antigen(s) driving determinant spreading might be derived from physiological apoptosis which could explain the involvement of some chaperone proteins in the autoimmune response. Diversified anti-La/Ro antibody responses were initiated by challenge with a single subdominant T epitope of La even though some self epitopes of La were efficiently tolerised. The pattern of autoantibody responses in primary Sjögren's syndrome was strongly influenced by HLA class II phenotype which we speculate controls activation of T cells recognising defined peptides from the La/Ro RNP. In this way, HLA class II alleles may be critical in influencing initiation and spreading of systemic autoimmune reactions. Molecular mimicry of such determinants by exogenous agents might readily initiate spreading of an autoimmune response in genetically susceptible hosts.

Amino Acid Sequence↗

PubMed1998

Spreading of the immune response from 52 kDaRo and 60 kDaRo to calreticulin in experimental autoimmunity.

Calreticulin (CR) is widely recognized as a new human autoantigen but there are conflicting data concerning its relationship with the Ro(SS-A) ribonucleoprotein (RNP). Recent evidence suggests that CR binds to 52 kDaRo (Ro52) by a protein/protein interaction and binds to hY RNA and rubella virus RNA. Other studies have shown that initiation of immunity to either Ro52 or 60 kDaRo (Ro60) can lead to reciprocal spreading of autoimmunity to Ro60 or Ro52, respectively, and induce anti-La autoantibodies in some strains of mice. These findings support a physical association of these polypeptides in Ro/La complexes. To test the hypothesis that CR is physically associated with Ro52 and/or Ro60 we examined the sera of Ro52-, Ro60- and La-immunized mice for intermolecular spreading to CR. Immune sera from BALB/c and C3H/HeJ mice immunized with recombinant 6xHis-mouse Ro52, 6xHis-human Ro60 or 6xHis-human La were tested for reactivity by ELISA and immunoblotting with a full-length human CR protein expressed as a soluble maltose binding protein fusion protein (CR-MBP). Five of the six Ro52-immunized C3H/HeJ mice sera and all six Ro60-immunized C3H/HeJ mice sera reacted with the CR-MBP (but not a MBP control) on ELISA. In the BALB/c group, the responder rate was lower with one in six of the Ro52-immunized and one in five of the Ro60-immunized mice spreading to CR. In contrast, none of the BALB/c or C3H/HeJ mice which was immunized with La showed evidence of a recruited anti-CR antibody response. Immunoblotting of the different recombinant proteins with immune sera from the C3H/HeJ mice confirmed the specificity of the initial and recruited antibody responses. The spreading of immunity from Ro52 and Ro60 to CR in Ro-immunized mice suggests that a subpopulation of CR or CR-like molecules must associate under certain circumstances with Ro52 and Ro60 polypeptides in vivo, possibly as Ro/CR complexes concentrated in surface membrane blebs of apoptotic cells. The lack of spreading to CR in La-immunized mice suggests that CR may be associated with a subpopulation of Ro particles from which La has already dissociated.

Animals↗

PubMed1997

Identification of residues in the class II-associated Ii peptide (CLIP) region of invariant chain that affect efficiency of MHC class II-mediated antigen presentation in an allele-dependent manner.

Invariant chain (Ii) associates with class II MHC molecules and is crucial for Ag presentation by class II molecules. A general explanation for how invariant chain (Ii) associates with polymorphic MHC class II molecules has been suggested by the crystallographic structure of CLIP (class II-associated Ii peptide) complexed with an HLA class II molecule, HLA-DR3. We show here that methionine residues at positions 93 and 99 in Ii are important in MHC class II-mediated Ag presentation, but function in an allele-dependent manner. Introduction of a Met-->Ala mutation at position 99 in Ii (M99AIi) impaired presentation of peptides derived from exogenous proteins by I-Ad and I-Au class II molecules. Mutating Met-->Ala in Ii at position 93 (M93AIi) abrogated presentation by I-Au molecules, but not by I-Ad. Impaired Ag presentation was associated with conformationally altered expression of I-A molecules on the surface of cells expressing mutated Ii. Cell surface CLIP staining and immunoprecipitation studies showed that both I-Ad and I-Au molecules were associated with an increased abundance of Ii peptides, CLIP, in cells expressing mutated Ii. These results show that methionine 93 and methionine 99 play an important physiologic role in Ii association with class II molecules by regulating release of CLIP from class II in the endocytic compartments to allow binding of cognate peptides.

Alanine↗

PubMed1997

Natural inactivation of a common HLA allele (A*2402) has occurred on at least three separate occasions.

HLA-A*2402 is common and widely distributed in human populations. Several individuals were identified who type genotypically for A*2402, but are serologically null for the HLA-A24 Ag. Sequencing and transfection of genomic DNA fragments containing null and wild-type A*2402 alleles, and the related A*2301 allele, revealed three different null alleles (A*2409N, A*2411N, and A*2402(low)), each of which differs from A*2402 by a single nucleotide change within the 6.7-kb sequence. The A*2301 and A*2402 sequences differ by no substitutions additional to those previously determined for the 1.1-kb cDNA. In exon 4, A*2409N has an in-frame stop codon, while A*2411N has a nucleotide insertion that alters the reading frame, causing premature termination. A*2402(low) has a nucleotide substitution near the splice acceptor site for intron 2 that impairs the production of correctly spliced mRNA. For A*2409N and A*2411N, mRNA is undetectable by Northern analysis, whereas A*2402(low) produces a low level of mRNA and a concomitant amount of normal A*2402 protein at the cell surface. The protein expressed from the A*2402(low) allele is sufficient to stimulate an alloreactive T cell response. On a background of unexpected sequence homogeneity, the single nucleotide changes in the A*2409N, A*2411, and A*2402(low) alleles have dramatic effects upon gene expression and are of likely importance for HLA matching in clinical transplantation. Segregation of at least three independently inactivated A*2402 alleles in human populations raises the possibility that loss of A*2402 may be the result of natural selection.

Alleles↗

PubMed1997

Human leukocyte antigen phenotype imposes complex constraints on the antigen-specific cytotoxic T lymphocyte repertoire.

The memory response to the immunodominant Epstein-Barr virus (EBV) epitope FLRGRAYGL, which associates with HLA B8, is exceptionally restricted, being dominated by cytotoxic T lymphocytes (CTL) with a single, public T cell receptor (TCR). CTL clones that express this receptor fortuitously cross-react with the alloantigen HLA B44. However, of the two major subtypes of this HLA, B*4402 and B*4403, that differ by a single amino acid, only the former is recognized by these mature CTL clones. Individuals heterozygous for HLA B8 and B*4402 use alternative TCR for the EBV determinant since the dominant TCR is potentially self-reactive. We now demonstrate that this clonotype is also essentially absent from the repertoire of CTL directed against the viral epitope in seven from seven unrelated individuals heterozygous for HLA B8 and B*4403. Thus immune tolerance of these CTL recognizing HLA B*4402 is associated with expression of either B*4402 or B*4403. This suggests that tolerance in the human T cell compartment requires a lower threshold of recognition than for effector function, thus providing a buffer zone minimizing the risk of autoimmunity. These data also illustrate the potential for non-restricting HLA molecules to bias dramatically the T cell repertoire used for specific immune responses. Such influences may be the basis of the "protective" effects of certain HLA alleles in susceptibility to autoimmune disorders.

Amino Acid Sequence↗

PubMed1997

Cross-reactive memory T cells for Epstein-Barr virus augment the alloresponse to common human leukocyte antigens: degenerate recognition of major histocompatibility complex-bound peptide by T cells and its role in alloreactivity.

In the present report, cytotoxic T lymphocyte (CTL) clones are described that display dual specificity for one of two common human leukocyte antigens (HLA B14 or B35) as alloantigens, and an immunodominant epitope (FLRGRAYGL) from Epstein-Barr virus (EBV) that binds to HLA B8. These T cell clonotypes were isolated from several unrelated HLA B8+, EBV-exposed individuals, and each distinct cross-reactivity pattern was associated with a common, public T cell receptor (TCR). In some individuals, CTL cross-reactive with these alloantigens completely dominated the memory response to this EBV epitope. Moreover, these memory T cells to EBV could be reactivated as a significant component of the repertoire of CTL responding to allogeneic stimulator cells expressing either HLA B14 or B35. These data illustrate how a history of infection with an immunogenic virus such as EBV can augment responsiveness to particular alloantigens; such influences may underlie the observed clinical association between herpesvirus infection and both allograft rejection and graft-versus-host disease. We have also explored the molecular basis for T cell cross-reactivity with alloantigens using the HLA B35 allo-reactive CTL clonotype. To elucidate the structural features of peptides that may be cross-recognized by these T cells, mono-substituted analogs of the viral epitope were screened for recognition, revealing broad specificity for major histocompatibility complex (MHC)-bound peptide. Based on the particular amino acid changes tolerated by the CTL at each peptide position, the human protein sequence database was searched for possible sequences that were recognized in association with HLA B35. Four peptides were identified (MPEATVYGL, IPIAPVYGM, KPSPPYFGL, and KPIVVLHGY) that were powerful activating ligands for the CTL when presented on HLA B35 but not B8. Thus, equivalent epitopes, capable of fully activating a single TCR, were formed by peptides with minimal obvious sequence homology bound to either HLA B8 or B35. These data indicate that degenerate peptide recognition by TCR may play an important role in the vigorous response of self-MHC-restricted T cells to alloantigens.

Antigen Presentation↗

PubMed1997

Platelet antigen allele frequencies in Australian aboriginal and Caucasian populations.

We have applied genotyping methods of PCR-SSOP and PCR-RFLP to three, bi-allelic platelet specific antigen systems HPA-1 (Pla), HPA-3 (Bak) and HPA-5 (Br). This combination of techniques offers flexibility for high volume or rapid typing. The phenotype and genotype frequencies of alleles from the three systems differ significantly between the Yuendumu Australian Aboriginals (Wailbri) and Australian Caucasians. The major differences are the very low frequencies of HPA-1b and HPA-3b in Yuendumu Aboriginals which are potentially relevant to platelet transfusion in patients of Australian Aboriginal descent.

Alleles↗

PubMed1997

Impaired antigen presentation by murine I-Ad class II MHC molecules expressed in normal and HLA-DM-defective human B cell lines.

The inability of certain antigen processing mutant cell lines to present intact proteins to T cells and to form SDS-stable MHC class II dimers has been shown to result from defective expression of HLA-encoded DMA and DMB genes. We have utilized some of these mutants to determine species compatibility of antigen presentation components. Mouse MHC class II I-Ad cDNA was transfected into the human B cell lymphoblastoid cell lines 8.1.6, 7.9.6 (a mutant cell line derived from 8.1.6) and an independent deletion mutant T2 (called 8.1.6d, 7.9.6d and T2.d respectively). These cells were than examined for various functions in antigen presentation. Interestingly, none of the cells transfected with I-Ad presented peptides derived from intact proteins to specific T cell hybridomas. However, presentation of synthetic peptides by these cells was normal. The ability to form SDS-stable dimers was dramatically reduced in the transfectants. In addition, I-Ad molecules at the cell surface appeared loaded predominantly with the invariant chain peptides, CLIP. These properties of the I-Ad transfectants are identical to those described for HLA class II molecules expressed in HLA-DM mutants. Perhaps the most interesting finding was the inability of I-Ad in 8.1.6 to present protein antigens. Since 8.1.6 cells present antigens to HLA-DR, DP, DQ-restricted T cells and also have intact HLA-DM and invariant chain (II) functions, these results argue that some component of human antigen processing machinery is incompatible with I-Ad molecules.

Animals↗

PubMed1997

Three newly identified A24 alleles: A2406, A2413 and A2414.

Three previously unknown A24-related alleles were identified by PCR-SSO typing and confirmed by DNA sequencing in Australian Aboriginal populations (A*2406, 2413) and in individuals of South American descent (A*2414). A*2406 and A*2413 both have two adjacent (but different) nucleotide substitutions in codon 156 in exon 3 compared to A*2402, resulting in a single amino acid replacement in each allele. The South American A*2414 is apparently a hybrid between A2 and A24 with a segment of the A*24 sequence between codons 95 and 107 in exon 3 replaced with the A*02 sequence. Interallelic sequence exchange is the most likely mechanism in the generation of all three novel alleles. Compared to A*2402, the four amino acid substitutions in the A*2414 molecule would be expected to significantly change the shape of the peptide binding cleft, leading to selection of different peptide ligands. The single amino acid replacements in position 156 of the two Australian Aboriginal A*24 alleles may also have significant functional effects. In particular, Trp replacing Gln in position 156 (A*2406) is predicted to markedly reduce the volume of the peptide binding cleft, influence the interaction of HLA pockets with peptide side chains, and therefore, cause major changes in peptide presentation. These newly defined alleles may reflect the adaptive process of HLA genes to local environments.

Alleles↗