A sensitive fluorometric assay for quantitatively measuring specific peptide binding to HLA class I and class II molecules

A sensitive, highly reproducible assay was developed for measuring binding of peptides to various HLA class I and II alleles. The assay is based on competition for binding to HLA between a peptide of interest and a fluorescent labelled standard peptide. This mixture is incubated with HLA to obtain equilibrium binding, and subsequently separated on an HPLC size-exclusion column in (i) a protein fraction containing HLA and bound peptide and (ii) a free peptide fraction. Each assay uses only 100 fmol labelled peptide and approximately 10 pmol of HLA. The analytical system contains an autosampler that samples from 96-well microtiter plates. Injections and data recording/evaluation is fully automated. Typical analysis time is 10–12 min per sample. The fluorescence in the HLA-bound peptide and free peptide containing fractions is measured on-line. The ratios of fluorescence signal in protein and peptide fractions at various concentrations of the peptide of interest are determined. IC₅₀ values are calculated from the binding curve as obtained by curve fitting of the data. Here we show results for peptide binding to HLA-DR1 and -DR17 molecules purified from detergent solubilized cell lysates, and for recombinant HLA-A^* 0201 and HLA-A^* 0301 expressed in E. coli.

The assay reported is sensitive and reproducible. It is non-radioactive and is non-labor intensive due to the high degree of automation.     

Predominant and stable T cell responses to regions of myelin basic protein can be detected in individual patients with multiple sclerosis

T lymphocytes from patients with multiple sclerosis (MS) recognize multiple myelin basic protein (MBP) epitopes. This situation complicates the design of specific immunotherapies. We investigated to which extent the T cell response to MBP is heterogeneous in single subjects in terms of preferentially recognized regions of the molecule, major histocompatibility complex (MHC) restriction, and stability over time. From each of nine patients with MS, a minimum of six MBP-specific T lymphocyte lines (TLL) were assayed for the proliferative response to a panel of overlapping peptides, encompassing the whole MBP. Predominant Tcell recognitions of distinct MBP regions were present in three patients, all HLA-DR2⁺, independently of the clinical features of their disease. Tcell reactivity was preferentially directed to residues 16-38 in one patient. In this case the response was also stable over time, during different phases of the disease. Predominant reactivity to residues 86-99 was detected in the two other DR2⁺ patients. In each of the patients with other HLA-DR haplotypes (DR2^?), as well as in three DR2⁺ non-MS donors, the Tcell response to MBP appeared to be considerably more heterogeneous. The HLA restriction element varied among TLL recognizing the same MBP region, even when raised from the same individual. The genomic HLA typing, performed on the DRB1 and DRB5 genes in the DR2⁺ subjects, showed no obvious correspondence between preferential responses to regions of MBP and HLA-DR2 subtypes. In this context, a simple, new method for the genomic typing of the HLA-DRB1 gene in individuals with the HLA-DR2 serological specificity is also described. We conclude that predominant and stable T cell responses to a single MBP region can be detected in some patients with MS. In these individuals, the MHC restriction of the T cell recognition of predominant regions appears to be variable. Polymorphisms of the HLA-DR2 gene products alone do not account for the selection of the dominant MBP Tcell epitope.     

口服髓鞘碱性蛋白诱导免疫耐受治疗实验性自身免疫性脑脊髓炎的研究

目的建立实验性自身免疫性脑脊髓炎（ＥＡＥ）动物模型,探讨口服自身抗原诱导免疫耐受对大鼠ＥＡＥ的防治作用。方法在普通Ｗｉｓｔａｒ大鼠经１次足跖真皮内注射完全弗氏佐剂－豚鼠全脊髓匀浆或完全弗氏佐剂－髓鞘碱性蛋白乳剂加百日咳疫苗诱发ＥＡＥ疾病;另在大鼠致炎前及发病后口服髓鞘碱性蛋白（ＭＢＰ）,观察其对ＥＡＥ的防治作用。结果在Ｗｉｓｔａｒ大鼠成功地诱发了ＥＡＥ,发病率将近９０％。大鼠在致炎前口服ＭＢＰ,可明显推迟ＥＡＥ发病时间,降低ＥＡＥ发病率,使神经组织病理改变明显改善。大鼠发生ＥＡＥ后给予ＭＢＰ,可明显控制发病动物的病情,使病程缩短及减轻患鼠神经组织的炎症程度。另外,在耐受鼠由ＭＢＰ引起的迟发型超敏反应（ＤＴＨ）以及体外针对ＭＢＰ的淋巴细胞增殖反应也明显受到抑制。结论口服ＭＢＰ可引起特异性的免疫耐受,从而产生对实验性自身免疫性脑脊髓炎的防治作用     

Spreading of the immune response to different myelin basic protein peptides in chronic experimental autoimmune encephalomyelitis in B10.RIII mice

B10.RIII mice develop chronic and relapsing experimental autoimmune encephalomyelitis (EAE) after immunization with the myelin basic protein (MBP) peptide 89-101 (VHFFKNIVTPRTP). To investigate the basis for the chronicity of the disease, the subsequent development of an immune responses to other parts of the MBP protein were investigated. Onset of disease occurs 9-25 days after immunization with MBP89-101. T cell responses towards a series of MBP peptides were assessed in an enzyme-linked immunospot assay detecting single cells secreting IFN-γ. There were responses not only to MBP89-101, but also towards peptides derived from sequences outside of MBP89-101. These peptides were of two kinds: those with sequences completely outside the 89-101 stretch of MBP; and those sharing a short sequence with MBP89-101 depending on alternative splicing of MBP mRNA. Immunization with these peptides also produced chronic EAE and a spreading of the immune response to other MBP peptides. Immunization with stepped peptides around the relevant region (MBP87-110) showed that peptides sharing a 6-amino-acid motif induced EAE after immunization. After MBP 89-101 peptide immunization, T cells isolated from lymph nodes did not cross-react in vitro to the other peptides sharing this motif. We suggest that one mechanism for the development of relapses during the disease course is the recruitment of new T cells with specificity for MBP peptides not derived from the peptide used for immunization. This is the first time such a mechanism has been demonstrated in a chronic autoimmune disease model.     

HLA class I and II polymorphisms in Azores show different settlements in Oriental and Central islands

Human leucocyte antigen-A, -B, -Cw, -DRB1, -DQA1 and -DQB1 polymorphisms were examined in the Azorean population. The data were obtained at high-resolution level, using polymerase chain reaction (PCR) with sequence-specific primer, PCR-sequence-specific oligonucleotides and sequence-based typing. The most frequent allele in each locus was: A*0201 (24.5%), B*510101 (9.8%), Cw*0401 (14.8%), DRB1*070101 (18.3%), DQA1*0201 (17.4%) and DQB1*0301 (19.4%). The predominant extended haplotype was A*0202-B*1503-Cw*0202-DRB1*090102-DQA1*0303- DQB1*0202 (1.9%), which was found to be absent in the Portuguese mainland. The present study corroborates historical sources that say the Azores were populated not only by Portuguese but also by other Europeans, mostly Flemish people. Despite dendrogram analysis showing some remote Asian genetic affinities, the lack of specific alleles and haplotypes from those populations does not allow us to conclude for direct influence. Haplotype and allele frequencies in Azores show no homogeneous distribution between Oriental and Central islands of this archipelago. The Oriental islands harbour several haplotypes already found in mainland Portugal and identified as Mediterranean and European. The Central group of islands on the contrary clearly shows an influence of north Europeans (most probably derived from a well-documented Flemish settlement), with much less affinity to mainland Portugal.     

Orally administered myelin basic protein in neonates primes for immune responses and enhances experimental autoimmune encephalomyelitis in adult animals

Antigen-driven tolerance is an effective method for suppression of autoimmune diseases. Adult animals can be tolerized against the induction of experimental autoimmune encephalomyelitis (EAE) by both oral and parenteral administration of myelin basic protein (MBP). We have found that in contrast to previous studies of neonatal tolerance in which parenterally administered autoantigens induced tolerance, the oral administration of MBP in neonatal rats did not result in tolerization to MBP, but instead, primed for immunologic responses. Proliferative responses to MBP and its encephalitogenic epitope were present in animals fed with MBP as neonates and co-culture of encephalitogenic T cells with cells from neonatal rats fed with MBP were associated with enhanced MBP responses rather than the suppression observed with cells from adult rats fed with MBP. Furthermore, neonates fed with MBP and immunized 6–8 weeks later with MBP in adjuvant to induce EAE revealed enhancement of disease severity, and were not protected from a second attack upon active reinduction of EAE. Subcutaneous injection of soluble MBP into neonates had no effect on EAE induction as adults, whereas intraperitoneal injection of MBP in neonates was associated with marked suppression of disease in adults. Suppression of EAE began to appear in animals fed with MBP at 4 weeks of age, and was similar to oral tolerance in adult animals when animals were fed at 6 weeks of age. These results suggest that immaturity of the immunoregulatory network associated with oral tolerance and sensitization to autoantigens via the gut in the neonatal period may contribute to the pathogenesis of autoimmune diseases.     

Serological and molecular analysis of HLA class I and II alleles in Thai patients with psoriasis vulgaris

Abstract: The HLA class I and class II alleles in 67 patients with type I psoriasis vulgaris, 23 patients with type II psoriasis vulgaris and 140 healthy individuals were analyzed. The frequencies of HLA-A2, -B46, -B57 and DQB1*0303 were significantly increased in type I psoriasis compared to the controls (Pc<0.05). Molecular analysis of HLA-A2 alleles showed an increase in HLA-A*0207 and a decrease in HLA-A*0203 in type I psoriasis. HLA-DQBl*0301 was significantly decreased in type I psoriasis compared to the normal controls (Pc<0.05). No association of any alleles with type II psoriasis was observed. This data demonstrated two susceptible haplo-types: HLA-A1-B57-DRB1*0701-DQA1*0201-DQB1*0303 (AH57.1) and HLA-A2-B46-DRB1*0901-DQA1*0301-DQB1*0303 (AH46.1) for type I psoriasis in the Thai population. Besides, the haplotype AH46.1 was also associated with type II psoriasis.     

Healthy monozygous twins do not recognize identical T cell epitopes on the myelin basic protein autoantigen

The T cell response against myelin basic protein (MBP) has been extensively studied in humans because of its putative role in the pathophysiology of multiple sclerosis (MS). Higher concordance rates in monozygous twins as well as an increased risk in relatives suggest the role of genetic factors in MS susceptibility. Very little is known about the shaping of T cell repertoire towards self antigens in humans and their contribution to disease susceptibility in autoimmune disorders. Here we report the comparative T cell epitope recognition patterns towards the MBP auto-antigen in healthy identical twins. We have established MBP-specific T cell lines from eight sets of twins and characterized their fine epitope specificity. Intra-pair comparison showed the co-existence of shared as well as distinct epitopes in six of eight pairs and a complete absence of concordant epitope recognition within two other pairs. These findings indicate that important differences in T cell repertoires against a self antigen may be observed between genetically identical healthy individuals, rendering difficult the interpretation of the differences which may be observed between identical twins discordant for an autoimmune disease.     

髓鞘碱性蛋白和肿瘤坏死因子α水平与Guillain-Barre综合征和多发性硬化的关系

目的 检测Guillain-Barre综合征(GBS)和多发性硬化(MS)患者血清髓鞘碱性蛋白(MBP)和肿瘤坏死因子α(TNF-α)的水平,探讨它们的相互关系.方法 采用ELISA法测定24例GBS和36例MS患者的血清MBP和TNF-α,并与28例其它神经系统疾病(OND)和30例健康对照(HC)进行比较.结果 GBS和MS组的MBP、TNF-α水平分别是(230±52),(236±56)和(200±46),(185±38)ng/L,均高于OND组及对照组(均P＜0.01),且GBS组的MBP、TNF-α水平高于MS组(P＜0.01).结论 MBP、TNF-α在Guillain-Barre综合征与MS发病中可能都起一定的作用,但在GBS中可能更突出.     

HLA-DR-restricted presentation of purified myelin basic protein is independent of intracellular processing

Antigen presentation to CD4⁺ T cells involves intracellular antigen processing and loading of peptides onto newly synthesized major histocompatibility complex (MHC)-class II molecules. Some antigens, such as the lipid-bound, native form of myelin basic protein (LB-MBP) can also be presented by recycling of cell surface MHC class II molecules. The data reported here demonstrate that a preparation of highly purified, delipidated MBP (HP-MBP) follows yet another presentation pathway. Similar to LB-MBP, presentation of HP-MBP to HLA-DR1-restricted T cells was independent of HLA-DM, of newly synthesized proteins, and of invariant chain expression. However, in contrast to LB-MBP, presentation of HP-MBP was also independent of internalization of surface HLA-DR molecules. The different requirements for the presentation of the two molecular forms of MBP were further confirmed by use of the protease inhibitor E64: presentation of LB-MBP but not of HP-MBP was inhibited after treatment of target cells with E64. Furthermore, intact HP-MPB bound to isolated HLA-DR molecules in vitro with an association rate that was considerably faster than that of short peptides. These results show that presentation of HP-MBP is independent of intracellular processing and suggest that it may be presented to T cells by direct binding to surface HLA-DR molecules.     

Soluble HLA class I antigen secretion by normal lymphocytes: relationship with cell activation and effect of interferon-gamma

HLA class I antigens are thought to be integral membrane proteins. However, soluble forms of these molecules have been detected. Our laboratory has recently shown that the predominant form of these soluble proteins present in human serum, spleen tissue and culture supernatant of activated lymphocytes exhibits molecular weight and structure similar to classical HLA class I antigens, but lacks HLA A or B polymorphic determinants. In the present study, the secretion of such soluble proteins by lymphocytes has been further explored. Phytohaemagglutinin-stimulated normal lymphocytes secrete considerable quantities of soluble HLA (sHLA) class I proteins. This secretion seems to be a general property of lymphocytes, since activation of T as well as B cells by appropriate mitogens equally induce sHLA I secretion. Lymphocytes require RNA and protein synthesis, but not DNA synthesis, for the secretion to occur. Kinetic studies reveal that maximal sHLA I secretion precedes the peak of DNA synthesis by 24 h. In vitro stimulation with antigens or alloantigens also provokes sHLA I secretion. Moreover, this phenomenon has also been detected for in vivo-activated lymphocytes, as enhanced spontaneous sHLA I secretion was observed in cultures of low-density blastic B and T cells, and of blood lymphocytes obtained from normal subjects who had received a booster immunization 5 days earlier. Interferon-gamma (IFN-gamma) increases the expression of membrane-bound class I antigens but does not induce any sHLA I secretion, suggesting that both molecules are under different regulatory mechanisms. Our results indicate that human lymphocytes, upon stimulation, actively secrete considerable amounts of a soluble form of these biologically relevant proteins.     

Expression of HLA class I-encoded cell surface antigens in transitional cell carcinoma of the urinary bladder

Abstract: I. Levin, B. Klein, S. Segal, A. Eyal, J. Gopas, J. Kaneti, M. Saadon, O. Kuperman. Expression of HLA class I-encoded cell surface antigens in transitional cell carcinoma of the urinary bladder.     

Mechanisms of loss of HLA class I expression on colorectal tumor cells

For several years this laboratory has studied the expression of HLA class I on established colorectal tumor cell lines and on fresh tumors. We review here the mechanisms by which colorectal tumor cells may lose surface expression of HLA class I molecules. Several independent mechanisms have been identified, including loss or mutations in β2-microglobulin genes, loss of HLA heavy chain genes, selective lack of expression of HLA alleles, and regulatory defects in HLA expression including loss of expression of the peptide transporters associated with antigen processing (TAP). The data suggest that colorectal tumor cells may evade tumor specific, HLA restricted immune attack by loss of HLA class I expression through a number of mechanisms.     

脊髓压迫性损伤神经纤维溃变和髓鞘碱性蛋白的表达变化

目的　观察脊髓压迫性损伤中白质神经纤维溃变及MBP的表达变化。 方法　采用自行设计的压迫装置制作脊髓压迫性损伤模型,运用luxol fast blue（LFB）、免疫荧光和western blot等方法来检测脊髓受压1、3、7 d后的白质纤维变化及MBP表达变化。 结果　脊髓受压后,白质髓鞘化神经纤维出现水肿,排列疏松、髓鞘缺失等退行性溃变;髓鞘化纤维数目逐渐减少。蛋白MBP表达随着压迫时间进行性下降。 结论　脊髓压迫性损伤可导致神经纤维溃变,MBP表达下调是神经纤维溃变的原因之一。     

HLA class I expression in bladder carcinomas

HLA class I molecules are frequently lost in a large variety of human carcinomas, possibly because of T-cell immune selection of major histocompatibility complex class I deficient tumor variants. We report that this phenomenon is also a frequent event in bladder carcinomas. Of a total of 72 bladder carcinomas, 72% of the tumors had at least one alteration in HLA class I expression. These altered HLA class I phenotypes were classified as total HLA class I loss (25%; phenotype I); HLA-A or/and HLA-B locus-specific loss (12%; phenotype III); and HLA class I allelic loss (35%; phenotype II or IV). Comparison of histopathological parameters with HLA class I expression showed a statistically significant relationship with the degree of differentiation and tumor recurrence.     

Transfected trophoblast-derived human cells can express a single HLA class I allelic product

The human trophoblast-derived JAR cell line, that does not express polymorphic HLA class I antigens even after IFN induction, can be stably transfected by genomic clones encoding the entire HLA-A2, -A3 and -B7 alpha-chain genes. The transfected genes were expressed at the cell surface in association with endogenous beta 2-microglobulin (shown by FCM analysis) as a single allelic product without reexpression of any endogenous class I gene (shown by 1D.IEF analysis). Furthermore, TNF-alpha and IFN-gamma, alone and synergistically, increase cell surface expression of transfected MHC class I/endogenous beta 2m heterodimers without induction of endogenous class I alpha-chain genes. These data show that the MHC class I-negative JAR human cell line might be used for transfections with the aim of establishing human cells expressing just one defined MHC class I allele for functional and regulatory studies. These findings are discussed in relation to the methylated status solely of endogenous class I alpha-chain genes in JAR cells and suggest that transfected class I genes are not regulated in the same fashion and, in particular, that constitutive and TNF/IFN inducible trans-acting regulatory factors able to bind to cis-promoter/enhancer sequences of class I DNA are likely to be present.     

Locus-specific amplification of HLA class I genes from genomic DNA: locus-specific sequences in the first and third introns of HLA-A, -B, and -C alleles

Abstract: We have identified locus-specific sequences in the first and third introns flanking the polymorphic second and third exons of HLA class I genes. PCR primers derived from these conserved sequences produced DNA fragments of the expected sizes for each of the HLA-A, -B, and -C loci in the amplification of genomic DNA. PCR products generated using each of the locus-specific sets of primers displayed exquisite locus specificity, as assessed by hybridization with oligonucleotide probes specific for ten classical and non-classical HLA class I genes. Amplification with these primer sets was effective and specific for the HLA alleles tested under the given PCR conditions. When hybridized with oligonucleotides derived from shared polymorphic sequence motifs, reaction patterns of PCR products from each locus were precisely as expected from published or database sequences. Chemiluminescent signals generated from digoxygenin-ddUTP-labeled probes were even for all samples and as strong as those obtained in MHC class II typing. These locus-specific primer sets derived from intron sequences provide an effective means to amplify genomic DNA which will facilitate PCR-based HLA class I typing methods. This will also allow HLA class I typing to be conducted with greater precision, at lower cost, and faster than previously described class I typing methodologies.