Mesoporous silicon microparticles enhance MHC class I cross-antigen presentation by human dendritic cells.

The mesoporous silicon microparticles (MSMPs) are excellent vehicles for releasing molecules inside the cell. The aim of this work was to use MSMPs to deliver viral specific MHC class I restricted epitopes into human antigen presenting cells (monocyte derived dendritic cells, MDDCs) to facilitate their capture, processing, and presentation to CD8+ (cytotoxic) T lymphocytes. We show for the first time that MSMPs vehiculation of antigenic peptides enhances their MHC class I presentation by human MDDCs to CD8 T lymphocytes.

Identification of a new mouse Cd1d2 allele.

The mouse CD1 system is formed by two closely related genes named Cd1d1 and Cd1d2. Cd1d1 encodes a molecule that presents antigens to NKT cells. The function of the Cd1d2 gene has not been elucidated. Here we described a method to analyse variations in mouse Cd1 genes. We found a new allele for Cd1d2 characterized by a point mutation, resulting in a replacement of alanine at position 176 by a valine.

Structural characterization of two CD1A allelic variants.

CD1 molecules are specialized in presenting lipidic antigens to T lymphocytes. They are structurally and evolutionary related to MHC molecules and show very limited polymorphism. We have previously described and partially characterized a new human CD1A allele differing from the wild type CD1A by a substitution of Cysteine by Tryptophan at position 52 in the alpha1 domain of the CD1A molecule. The frequency of this allele varies from 10% in individuals of Caucasian origin to 56% in Chinese people. The aim of the present work was to structurally characterize this CD1A allele. To do this we have cloned and sequenced the full-length cDNA encoding the new CD1A allele. The cDNA sequence of this allele encodes a protein differing the wild type in two amino acids at positions 14 (Threonine versus Isoleucine) and 52 (Cysteine versus Tryptophan). The cDNAs encoding both wild type and mutant CD1A were cloned in the expression vector pSRalphaNeo and transfected into C1R and L721.221 cells. Cell surface expression of the protein products in transfected cell lines were analyzed by flow cytometry and immunoprecipitation using CD1a-specific monoclonal antibodies. Our results indicate that both allelic products are efficiently expressed on the cell surface.

Estructura y función de las moléculas HLA de clase I "No clásicas" / Structure and function of "non-classical" HLA class I molecules

En los últimos años se han descubierto diversos genes relacionados con los que codifican las moléculas HLA de clase I a los que se ha denominado en conjunto genes HLA "no clásicos". Hasta el momento se han descrito 9 familias "no clásicas" diferentes que contabilizan un total de 17 genes funcionales. Solamente para una de estas familias, MR1, la función sigue siendo completamente desconocida. Además, la estructura tridimensional de al menos una molécula representativa ha sido descifrada en 6 familias. Aunque muestran importantes diferencias en términos de secuencia aminoacídica las distintas moléculas HLA de clase I no clásicas presentan una estructura tridimensional muy parecida. Sorprendentemente, estas moléculas tan parecidas en su forma llevan a cabo funciones muy heterogéneas que van desde la presentación de antígenos lipídicos a los linfocitos T hasta la regulación del metabolismo del hierro (AU)

Characterization of the MHC class I-related MR1 locus in nonhuman primates.

We characterized the MHC-related 1 ( MR1) locus in two nonhuman primates species, Pongo pygmaeus and Pan troglodytes. MR1 cDNA sequences encoding several isoforms generated through alternative splicing were observed in both species. Amino acid alignment between the five species in which MR1 has been characterized to date - human, chimpanzee, orangutan, mouse, and rat - reveals a very high degree of conservation specially in the alpha1 and alpha2 domains of the molecule. The main differences concentrate in the transmembrane and cytoplasmic domains. In the three primates species there is a lysine residue inside the putative transmembrane domain which is not present in rodents. Furthermore, the MR1 cytoplasmic region is longer in rodents, with a conserved serine-containing motif that could be involved in endocytosis; remarkably, this motif is absent in the three primate species. We also describe the presence in the chimpanzee of a sequence homologous to the MR1P1 pseudogene previously found in humans.

Identification of two novel human CD1E alleles.

CD1 is a family of proteins structurally related to major histocompatibility complex (MHC) molecules and specialized in presenting lipids or glycolipids to T cells. In humans, there are five CD1 genes (CD1A to CD1E). It has been shown that, in contrast with classical MHC genes, CD1 loci display a very limited polymorphism. In the present work we describe two novel CD1E alleles found in two healthy Caucasian individuals. One allele differs from the wild-type by a point mutation resulting in a replacement of arginine at position 154 by a tryptophan. In the second allele we found a substitution of the leucine 184 by a proline.

A study on the polymorphism of human MHC class I-related MR1 gene and identification of an MR1-like pseudogene.

Human MR1 is a recently discovered, ubiquitously transcribed gene very similar to the HLA class I loci and of unknown function. Mouse and rat MR1 sequences have also been described showing high similarity with the human gene. The goal of this work was to investigate if human MR1 was polymorphic. We have found that DNA sequences of MR1-specific polymerase chain reaction (PCR) products obtained from samples of diverse ethnic origin were invariant except in one case in which two silent mutations were detected. We also found an MR1-like sequence displaying significant differences with the previously described, the most remarkable of which is a STOP codon in the alpha2 domain indicating that is a pseudogene.

Single strand conformational polymorphism analysis of human CD1 genes in different ethnic groups.

CD1 molecules are able to present unusual antigens, lipids or glycolipids from mycobacterium cell walls to T lymphocytes. Previous studies have suggested that polymorphism of these genes is very limited, in contrast with classical major histocompatibility complex (MHC) antigen-presenting molecules. Our aim was to study possible allelic variations of exons 2 and 3, encoding for the alpha1 and alpha2 domains, respectively, of human CD1A, -B, -C and -D genes. We analyzed genomic samples of unrelated, healthy individuals from different ethnic background: 70 Caucasians from Europe, 33 Black Africans (13 from Tanzania and 20 Zulus), 19 Caucasians from the Sahara and 44 Asian individuals. We have found CD1A to be a biallelic locus with a common allele which was present in the majority of the individuals studied. The second allele differed from the common one by a single-point mutation, resulting in a change of Cys to Trp at position 52 in the alpha1 domain. This second allele was found in heterozygosis in 7 out of 70 Caucasians from Europe (allelic frequencies P=0.95 and q=0.05). In the Chinese population, we found the second allele present in heterozygosis in 19 from the 44 individuals studied, and we also found 6 homozygous individuals for the second allele (allelic frequencies P=0.64 and q=0.35). In addition, we detected a synonymous mutation (C to T transition) in codon 34 of CD1C exon 2 in 4 out of 20 Zulus and in 2 of the 13 Blacks from Tanzania.

Interactions of HLA-B*4801 with peptide and CD8.

Functional properties of the B*4801 allotype were investigated using HLA class I-deficient 221 cells transfected with B*4801 cDNA. From pool sequence analysis of endogenously bound peptides, B*4801 was shown to select for nonamer peptides having glutamine or lysine at position 2 and leucine at the carboxyl-terminus. In an in vitro cell-cell binding assay, B*4801 binds CD8 alpha homodimers weakly due to the presence of a threonine residue at position 245 in the alpha 3 domain. A mutant B*4801 molecule in which alanine replaces threonine 245, binds CD8 alpha homodimers at levels comparable to those of other HLA class I allotypes. Despite the low affinity of B*4801 for CD8 alpha, alloreactive T-cells that recognize B*4801 molecules expressed by the 221 transfectant are inhibited by anti-CD8 monoclonal antibodies. Analysis of 25 B*48-expressing individuals from various populations showed threonine 245 was encoded by every B*48 allele.

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.

HLA-B16 antigens: sequence of the ST-16 antigen, further definition of two B38 subtypes and evidence for convergent evolution of B*3902.

The ST-16 antigenic specificity of the HLA-B locus is defined as a B39 variant of Mexican-Americans. Nucleotide sequencing of cDNA shows the ST-16 allele (B*3905) differs from B*39011 by a single substitution that substitutes tyrosine for aspartic acid at position 74 of the mature class I heavy chain. The complete coding region sequence for the common caucasoid allele encoding the B38 antigen has been determined. This B*3801 allele differs from B*3802 at two nucleotide substitutions within the Bw4 sequence motif. B*3801 and B*3802 may have been derived independently from B*39011 by conversion events with B alleles donating distinctive Bw4 motifs. A novel allele B*39022 derived from a Colombian Indian differs from the B*39021 allele of Japanese origin at two widely separated silent substitutions. Comparison of sequences for the known B16 alleles suggest that B*39021 and B*39022 were independently derived by recombination from B*39013 and B*39011 respectively.

HLA class II and susceptibility and resistance to insulin-dependent diabetes mellitus in a population from the northwest of Spain.

The role of HLA class II alleles in genetic predisposition to insulin-dependent diabetes mellitus (IDDM) was examined using Polymerase Chain Reaction/oligonucleotide probe typing (PCR/SSOs) of eight HLA class II loci in 58 IDDM patients and 50 healthy controls from the Northwest of Spain (Asturias). We compared the distribution of HLA class II alleles, haplotypes and genotypes between IDDM patients and controls, and tested three recently proposed HLA-IDDM susceptibility theories. By using the aetiologic fraction (delta) as an almost absolute measure of the strongest linkage of disequilibrium of a HLA marker to the putative Type I susceptibility locus, it has been found that the strength of association of the HLA markers may be quantified as follows: DQA1*03-DQB1*0302 or DQA1*0501-DQB1*0201 > DR3 or DR4; presence of more than one dimer DQ alpha beta of the six proposed by Rønningen > non-Asp57 DQ beta and Arg52 DQ alpha > Arg52 DQ alpha > non-Asp57 DQ beta/non-Asp57 DQ beta > DRB1*0301; DQA1*0501-DQB1*0201 > DQA1*03-DQB1*0302; DQB1*0302. The presence of at least one Asp57 DQ beta allele was the best protection HLA marker to IDDM in our population. Therefore, the above data confirm that IDDM susceptibility to HLA locus is linked more to DQ than DR.

T-cell receptor alpha, delta, and gamma chain genes in insulin-dependent diabetes mellitus.

We have studied the genotypic, haplotypic, and allelic distribution of germline Restriction Fragment Length Polymorphism (RFLP) of T-cell receptor (Tcr) alpha, gamma, and delta loci in 75 insulin-dependent diabetes mellitus (IDDM) patients and 84 healthy blood donors as control population. The restriction endonuclease PvuII produces three allelic fragments of Tcr C gamma (TcrCG) gene segment of 16, 13, and 11.3 Kb respectively. Our observations revealed that PvuII/TcrCG RFLP allelic distributions were not significantly different in the IDDM and the control group. However, 85% of IDDM patients carried HLA DR3 and/or DR4 haplotypes, and when comparing these patients with a second group of HLA DR3+ and/or DR4+ healthy individuals, the 11.3 Kb/PvuII fragment of TcrCG gene was found to be associated with IDDM patients (chi 2 = 11.4, P = 0.003). 54.9% of IDDM patients carried at least one 11.3 Kb allele vs. 21% in controls (chi 2 = 10.77, P = 0.004). No significant association was found between RFLP in Tcr, C alpha, C delta, V gamma 9 loci and IDDM.

The germline repertoire of T cell receptor beta-chain genes in multiple sclerosis patients from Spain.

Recently several reports have described contradictory results after studying the association between restriction fragments length polymorphisms (RFLP) of T cell receptor (TcR) beta-chain genes and multiple sclerosis (MS). We studied the allelic, genotypic and haplotypic distribution of RFLPs of TcR beta chain gene segments C beta, V beta 8 and V beta 11 in 97 unrelated multiple sclerosis (MS) patients, 11 with chronic progressive MS and 86 with relapsing/remitting (R/R) MS. We found the distribution of the TcR haplotypes defined by the alleles of the three loci studied in the MS patients was significantly different from that found in control individuals. The distribution of TcR haplotypes in R/R MS patients was also different from that observed in controls. Our data suggest that the TcR beta chain gene complex contains one or more genes involved in genetic susceptibility to develop MS.

Molecular typing of HLA-B27 alleles.

HLA-B27 represents a family of closely related antigens. Six alleles which differ in a limited number of nucleotide substitutions have been described (B*2701--B*2706). These changes are clustered in alpha 1 and alpha 2 domains. Polymerase chain reaction strategies were designed to amplify specific regions of class I exons 2 and 3. Amplified sequences were tested with eight sequence-specific oligonucleotides to distinguish all B27 subtypes. We also subtyped B27 in 50 healthy Spanish individuals using this procedure. The B*2705 subtype is over-represented in our population (96%). The remaining 4% carried the B*2702 allele. This finding is in agreement with the frequencies described by other techniques (cytotoxic T lymphocytes and isoelectric-focusing) for Caucasian populations. Class I oligotyping is a poorly developed field with significant potential applications. This procedure of genotyping B27 alleles is a reliable method which can be used in transplantation and B27-associated disease studies.

DNA analysis of HLA-DR4B1 subtypes in multiple sclerosis by specific oligonucleotide probes.

Conversely to the well-established association of DR2/Dw2 with multiple sclerosis (MS) susceptibility in Caucasoids, several studies have found an association of DR4 in populations from Mediterranean origin. We have studied the distribution of the different DR4B1 subtypes in Spanish MS patients. Oligonucleotide probes were selected in order to type samples amplified by polymerase chain reaction (PCR) from Spanish DR4+ MS patients (25) and controls (28). No DR4B1 subtypes were found to be increased in MS. MS susceptibility linked to DR4 may be due to the presence of shared functional epitopes common to the different HLA-DR4B1 subtypes.

Germline repertoire of T-cell receptor beta-chain genes in patients with insulin-dependent diabetes mellitus.

We have investigated the genotype and allelic distribution of germline restriction fragment length polymorphisms of the T-cell receptor beta chain, segment C beta, and two variable segments which are in linkage disequilibrium, V beta 8 and V beta 11, in 42 insulin-dependent diabetes mellitus (IDDM) patients and in 51 healthy blood donors used as controls. Recently, several works have reported contradictory results showing or not showing an association between polymorphic alleles of the C beta gene and diabetes type I. We found no significant differences in the allele, genotype, and haplotype distribution of the gene segments studied, between IDDM patients and control populations.