Identification of a new candidate locus for uric acid nephrolithiasis.

Renal stone formation is a common multifactorial disorder, of unknown etiology, with an established genetic contribution. Lifetime risk for nephrolithiasis is approximately 10% in Western populations, and uric acid stones account for 5%-10% of all stones, depending on climatic, dietary, and ethnic differences. We studied a small, isolated founder population in Sardinia, characterized by an increased prevalence of uric acid stones, and performed a genomewide search in a deep-rooted pedigree comprising many members who formed uric acid renal stones. The pedigree was created by tracing common ancestors of affected individuals through a genealogical database based on archival records kept by the parish church since 1640. This genealogical information was used as the basis for the study strategy, involving screening for alleles shared among affected individuals, originating from common ancestors, and utilization of large pedigrees to obtain greater power for linkage detection. We performed multistep linkage and allele-sharing analyses. In the initial stage, 382 markers were typed in 14 closely related affected subjects; interesting regions were subsequently investigated in the whole sample. We identified two chromosomal regions that may harbor loci with susceptibility genes for uric acid stones. The strongest evidence was observed on 10q21-q22, where a LOD score of 3.07 was obtained for D10S1652 under an affected-only dominant model, and a LOD score of 3.90 was obtained using a dominant pseudomarker assignment. The localization was supported also by multipoint allele-sharing statistics and by haplotype analysis of familial cases and of unrelated affected subjects collected from the isolate. In the second region on 20q13.1-13.3, multipoint nonparametric scores yielded suggestive evidence in a approximately 20-cM region, and further analysis is needed to confirm and fine-map this putative locus. Replication studies are required to investigate the involvement of these regions in the genetic contribution to uric acid stone formation.

Modulation of TCR recognition of MHC class II/peptide by processed remote N- and C-terminal epitope extensions.

N- and C-terminal extensions of naturally processed MHC class II-bound peptides may affect TCR recognition. In fact, residues immediately flanking the minimal epitope on either side can contact the MHC groove and modify the interaction with a TCR. We report now that residues much farther away from the peptide core can also modulate TCR recognition in a functional antigen presentation system. To show this, we isolated from the same donor DR5-restricted T cell clones, specific for the HIV-1 RT(248-262) sequence and differing in their ability to respond to recombinant antigens obtained by insertion of the epitope in different positions of schistosomal, human, or murine glutathione-S-transferase (GST). We found that the reactivity profile of individual clones was related to their TCR fine specificity, suggesting that processing can generate determinants focused onto the same epitope, but antigenically distinct. In addition, we analyzed the response of this panel of T-helper cell clones against GST-derived recombinant antigens in which the epitope was flanked by stretches of polyalanine or polyserine on either side. These spacers had different effects on TCR recognition suggesting that secondary structures outside the core peptide may influence MHC/epitope complex recognition over a distance of 15-30 residues from the determinant.

In vitro immunization with a recombinant antigen carrying the HIV-1 RT248-262 determinant inserted at different locations results in altered TCRVB region usage.

Immunodominance or cripticity of a peptide-borne determinant may be influenced by the protein context in which the epitope is embedded. In this frame, we previously showed that certain human T cell clones, derived from different donors, may differentially recognize the RT248-262 helper determinant depending on whether it is provided to the presenting cells as a synthetic peptide or as a recombinant carrier protein to which the sequence of interest is fused. We now report that, upon in vitro immunization of human PBL with autologous APC, the epitope-specific TCRVB repertoire obtained when selection is applied by pulsing the APC with the cognate synthetic peptide is different from that found when a recombinant protein is used in which the antigenic sequence is placed at either a N-terminal or C-terminal location of the GST carrier. As the TCRVB distribution is not a function of the APC used, we propose that processing of different recombinant molecules containing the same epitope may generate MHC/peptide complexes which, being antigenically diverse, may recruit distinct TCR specificities. These findings may be relevant for evaluating and predicting the immunogenic potential of subunit vaccines based on synthetic peptides or on recombinant proteins as compared to the native antigen.

Antagonistic activity of HIV-1 T helper peptides flanked by an unrelated carrier protein.

Antagonism is the ability of a modified antigenic peptide (altered peptide ligand, APL) to prevent CD4 T cell activation by the original peptide. Here we show that antagonistic activity can be conferred to peptides of HIV envelope glycoprotein gp120 and reverse transcriptase p66 by adding flanking polypeptide sequences at the C or at the N terminus by genetic engineering, rather than by introducing substitutions by synthesis. The glutathione S-transferase (GST)-peptide system has been used to produce molecules that display the peptide at the appropriate end of the GST carrier. When the gp120 peptide 191-205 (pep24) was expressed at the C terminus of GST (GST-24), antigenicity of specific human CD4 T cells was maintained. In contrast, when the peptide was expressed at the N terminus of GST (24-GST), antigenicity was abolished and antagonistic activity was introduced. Similar results were obtained with a p66-derived peptide at the C terminus of the GST carrier. Antagonism was (1) specific; proliferation of a CD4 T cell line from the same donor responding to the envelope glycoprotein of another retrovirus, HTLV-1, was not affected; (2) reversible; proliferative response was rescued in T cells exposed to antigen-presenting cells (APC) pulsed with the antagonist; (3) dominant; T cells cultured with APC pulsed with the agonist and with APC pulsed with the antagonist did not proliferate. The carrier could be cleaved by proteolysis while the antagonistic activity was preserved. Thus a minimal sequence that confers antagonistic activity can be engineered or synthesized with peptides to antagonize undesired CD4 responses as an alternative to the use of APL.

Recognition of HIV-derived B and T cell epitopes displayed on filamentous phages.

The amino acid sequence of HIV reverse transcriptase (RT) from residue 248 to residue 262 was expressed on the surface of filamentous phage fd, fused to the major coat protein gVIIIp. The chimeric phage was used to assess the ability of anti-RT (248-262) human T cell lines and clones to become activated by the phage-displayed peptide. The RT peptide displayed on phage was recognized by the T-cells and induced production of Abs. However, not all T cells raised against the synthetic RT (248-262) peptide could respond. Lack of recognition did not depend on differences in the ability of different APCs to present the phage, but was apparently determined by the TCR specificity. The results presented here may be relevant to the design of recombinant protein-based subunit vaccines.

Antigenicity of HIV-derived T helper determinants in the context of carrier recombinant proteins: effect on T helper cell repertoire selection.

T helper (Th) epitopes can be included in a recombinant protein with B and CTL epitopes to create more effective immunogens. To determine whether the antigenicity of HIV Th epitopes is preserved in this altered molecular context, human Th clones specific for peptides of HIV gp120 and reverse transcriptase p66 were challenged with recombinant proteins carrying the antigenic epitopes in different sites. We found that a given epitope was recognized by a specific T cell clone only when it was inserted in a particular position of the carrier. However, the permissive position was not the same for all epitopes. Enzymatic excision from a nonpermissive context or insertion of a polyserine spacer between the epitope and the carrier restored antigenicity. Nevertheless, antigenicity was not abolished in a synthetic peptide encompassing the epitope and the neighboring residues from the nonpermissive location. These data suggest that, in this case, the primary sequence of the chimeric protein flanking the HIV peptide is not responsible for loss of antigenicity. Furthermore, constructs carrying the epitope in a given position were recognized by peptide-specific Th clones raised from some individuals, but not from others. We show that this is due neither to individual modes of processing nor to the use of distinct major histocompatibility complex MHC class II restriction elements, but rather that it is related to the fine specificity of the clones. To study the effect of epitope context on selection of T cell repertoire in a naive individual, T cell lines were generated in vitro by stimulation with different peptide constructs. This resulted in the induction of diverse clonotypes defined by the pattern of recognition of different constructs, by T cell receptor V beta gene usage and by fine epitope mapping.

Long-term culture and T cell receptor analysis of T cell clones isolated from a patient with adenosine deaminase deficiency and type I diabetes.

We performed limiting dilution culture of T cells from a patient affected by primary immunodeficiency as a result of complete lack of adenosine deaminase (ADA) activity and also affected by insulin-dependent diabetes mellitus (type I diabetes). Despite the occurrence of immunodeficiency, we were able to raise and grow T cell clones derived from this patient in long-term culture. These T cells displayed ADA enzymatic activity and produced interleukin-2 after engagement of their T cell receptor (TCR)/CD3 complex. We analyzed the TCR repertoire of such clones by nucleotide sequencing of TCR beta chains. The results show that the T cell clones express different V beta but similar J regions. However, the CDR3 regions which are implicated in antigen recognition were found to be heterogeneous.

Reversion of a transcriptionally defective MHC class II-negative human B-cell mutant.

RJ2.2.5, a mutant derived from the human B-lymphoma cell, Raji, is unable to express the MHC class II genes because of a recessive transcriptional defect attributed to the lack of an activator function. We report the isolation of a RJ2.2.5 revertant, namely AR, in which the expression of the mRNAs encoded by these genes is restored. Comparison of the binding of nuclear extracts or of partially purified nuclear preparations from the wild-type, the mutant and the revertant cells to a conserved MHC class II promoter element, the X-box, showed no alteration in the mobility of the complexes thus formed. However, in extracts from RJ2.2.5, and other MHC class II negative cell lines, such as HeLa, the amount of complex observed was significantly higher than in wild-type Raji cells. Furthermore, the binding activity exhibited by the AR revertant was lower than that of the RJ2.2.5 and higher than that of Raji. The use of specific monoclonal antibodies indicated that in all cases c-Jun and c-Fos or antigenically related proteins were required for binding. An inverse correlation between the level of DNA-protein complex formed and the level of MHC class II gene mRNA expressed in the three cell lines was apparent, suggesting that overexpression of a DNA binding factor forming complexes with class II promoter elements may cause repression of MHC class II transcription. A model which reconciles the previously ascertained recessivity of the phenotype of the mutation carried by RJ2.2.5 with the findings reported here is discussed.

DNA polymorphisms in the 5'-flanking region of the HLA-DQA1 gene.

The HLA-DQA1 gene exhibits haplotype-specific restriction fragment polymorphisms due to DNA rearrangements. We found that some of these polymorphisms extend into the 5' flanking region of the gene and are distinct from other HLA-DQA1 related DNA polymorphisms so far reported. Sequencing of genomic DNA subclones derived from the 5' flanking region of HLA-DQA1 showed the presence, in a DR4 haplotype, of two repetitive elements of the Alu family, oriented in opposite directions and bracketing an approximately 3 kilobase region immediately adjacent to the promoter of the gene. When DNAs extracted from several cell lines were analyzed by genomic hybridization using single-copy probes relative to these intervening sequences, polymorphisms were observed. No structural alterations of the gene immediately outside the DNA portion delimited by the two Alu elements were observed, thus suggesting that polymorphisms of the 5' end of HLA-DQA1 may be limited to the intervening region between the two Alu repeats. The latter includes upstream regulatory elements controlling the expression of the genes. The possibility that the structure of the DNA in this region may influence the regulation of HLA-DQA1 gene expression in different haplotypes is discussed.

Effect of the AIR-1 locus on the activation of an enhancerless HLA-DQA1 promoter.

Studies on the regulation of a major histocompatibility complex (MHC) class II gene, HLA-DQA1, in Ia-positive cells (Raji, a human B-lymphoma cell line) and in isogenic Ia-negative cells (RJ2.2.5, a mutant of Raji altered at the AIR-1 locus) are reported. As previously found, AIR-1 is required in its entirety for the activity of an enhancer factor, the absence of which abolishes transcription of MHC class II genes. In this paper, we show that HLA-DQA1 gene expression can be directed by an enhancerless promoter. The fact that this promoter is inactive in the RJ2.2.5 mutant suggests that the trans-acting element determined by the AIR-1 locus is not only an enhancer factor as previously described, but also acts at the MHC class II promoter level.