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Results for “Positive selection”

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PubMed1997

In vitro positive selection of alpha beta TCR transgenic thymocytes by a conditionally immortalized cortical epithelial clone.

Development of mature CD4 and CD8 single-positive T cells requires a process known as positive selection, which depends on the specific recognition of self-peptide-MHC complexes on thymic stromal cells by immature CD4+CD8+ thymocytes. We have used an in vitro reaggregate system to study the positive selection of thymocytes by conditionally immortalized thymic epithelial clones. Thymocytes from mice transgenic for the F5 alpha beta TCR, specific for a peptide from the influenza nucleoprotein in the context of H-2Db, are positively selected in the H-2b MHC background, but fail to mature in mice expressing the H-2q haplotype. Development of embryonic day 15 F5 H-2q transgenic thymocytes was followed in reaggregate cultures supplemented with H-2b-expressing epithelial clones. A conditionally immortalized cortical epithelial clone, derived from H-2Kb-tsA58 transgenic mice, was found to be as efficient as freshly isolated thymic stromal cells in positively selecting CD8 transgenic thymocytes. In contrast, an H-2b-expressing kidney epithelial clone did not augment positive selection above background levels, implying that the effect of the thymic epithelial clone was not merely the presentation of selecting MHC molecules. Mature transgenic thymocytes generated in reaggregate cultures were able to differentiate into functionally competent cytotoxic T cells. This model provides an important in vitro system for the detailed study of the specific molecular interactions leading to positive selection of developing thymocytes.

Animals↗

PubMed2003

Tests for positive selection on immune and reproductive genes in closely related species of the murine genus mus.

We examine variation among species of Mus in four genes involved in reproduction and the immune response for evidence of positive selection: the sperm recognition gene Zp-3, the testis-determining locus Sry, the testicular cell surface matrix protein Tcp-1, and the immune system protein beta(2) m. We use likelihood ratio tests in the context of a well-supported phylogeny to determine whether models that allow for positively selected sites fit the sequences better than models that assume purifying selection. We then apply a Bayesian approach to identify particular sites in each gene that have a high posterior probability of being under positive selection. We find no evidence of positive selection on the Tcp-1 gene, but for Zp-3, Sry, and beta(2) m, models that allow for positively selected sites fit the sequences better than alternatives. For each of these genes, we identify sites that have a high (> 95%) posterior probability of being positively selected. For Zp-3, two of these sites occur near the sperm-binding region, while one occurs in a region whose functional role remains unstudied but where the pattern of change predicts functional importance. A single site in Sry shows an elevated rate of replacement substitution but occurs in a region of apparently little functional importance; therefore, relaxation of functional constraints may better explain the rapid evolution of this site. Three sites in beta(2) m have a posterior probability > 50% of being under positive selection. While the functional role for two of these sites is unknown, the third is known to influence the ability of MHC class I molecules to present antigens to the immune system; therefore, the elevated rate of replacement substitutions at this site is consistent with selection acting to promote variability in immune system proteins.

Animals↗

PubMed1996

Low synonymous site variation at the lacY locus in Escherichia coli suggests the action of positive selection.

We have determined the nucleotide sequences of seven lacY alleles isolated from natural isolates of Escherichia coli. Nucleotide heterozygosity estimates for this locus were compared to those obtained from previous studies of intraspecific variation at chromosomal loci, revealing that lacY has unusually low synonymous site variation. The average pairwise heterozygosity of synonymous sites (Ks = 0.0112 +/- 0.0100) is the second lowest reported and the lowest for loci that have an equivalent level of nonsynonymous variation. We consider several hypotheses to explain how different forces in evolution could act to create the observed pattern of polymorphism, including selection for translational efficiency and positive selection. Our analysis most strongly supports the hypothesis that positive selection has acted on the lacY locus in E. coli.

Alleles↗

PubMed2001

An algorithm for mapping positively selected members of quasispecies-type viruses.

BACKGROUND: Many RNA viruses do not have a single, representative genome but instead form a set of related variants that has been called a quasispecies. The sequence variability of such viruses presents a significant bioinformatics challenge. In order for the sequence information to be understood, the complete mutational spectrum needs to be distilled to a biologically relevant and analyzable representation. RESULTS: Here, we develop a "selection mapping" algorithm--QUASI--that identifies the positively selected variants of viral proteins. The key to the selection mapping algorithm is the identification of particular replacement mutations that are overabundant relative to silent mutations at each codon (e.g., threonine at hemagglutinin position 262). Selection mapping identifies such replacement mutations as positively selected. Conversely, selection mapping recognizes negatively selected variants as mutational "noise" (e.g., serine at hemagglutinin position 262). CONCLUSION: Selection mapping is a fundamental improvement over earlier methods (e.g., dN/dS) that identify positive selection at codons but do not identify which amino acids at these codons confer selective advantage. Using QUASI's selection maps, we characterize the selected mutational landscapes of influenza A H3 hemagglutinin, HIV-1 reverse transcriptase, and HIV-1 gp120.

Algorithms↗

PubMed1995

T cell receptor-mediated signaling events in CD4+CD8+ thymocytes undergoing thymic selection: requirement of calcineurin activation for thymic positive selection but not negative selection.

The goal of this study was to identify the differences of intracellular signals between the processes of thymic positive and negative selection. The activation of calcineurin, a calcium- and calmodulin-dependent phosphatase, is known to be an essential event in T cell activation via the T cell receptor (TCR). The effect of FK506, an inhibitor of calcineurin activation, on positive and negative selection in CD4+CD8+ double positive (DP) thymocytes was examined in normal mice and in a TCR transgenic mouse model. In vivo FK506 treatment blocked the generation of mature TCRhighCD4+CD8- and TCRhighCD4-CD8+ thymocytes, and the induction of CD69 expression on DP thymocytes. In addition, the shutdown of recombination activating gene 1 (RAG-1) transcription and the downregulation of CD4 and CD8 expression were inhibited by FK506 treatment suggesting that the activation of calcineurin is required for the first step (or the very early intracellular signaling events) of TCR-mediated positive selection of DP thymocytes. In contrast, FK506-sensitive calcineurin activation did not appear to be required for negative selection based on the observations that negative selection of TCR alpha beta T cells in the H-2b male thymus (a negative selecting environment) was not inhibited by in vivo treatment with FK506 and that there was no rescue of the endogenous superantigen-mediated clonal deletion of V beta 6 and V beta 11 thymocytes in FK506-treated CBA/J mice. DNA fragmentation induced by TCR activation of DP thymocytes in vitro was not affected by FK506. In addition, different effects of FK506 from Cyclosporin A on the T cell development in the thymus were demonstrated. The results of this study suggest that different signaling pathways work in positive and negative selection and that there is a differential dependence on calcineurin activation in the selection processes.

Animals↗

PubMed1994

T cell receptor antagonist peptides induce positive selection.

We have used organ culture of fetal thymic lobes from T cell receptor (TCR) transgenic beta 2M(-/-) mice to study the role of peptides in positive selection. The TCR used was from a CD8+ T cell specific for ovalbumin 257-264 in the context of Kb. Several peptides with the ability to induce positive selection were identified. These peptide-selected thymocytes have the same phenotype as mature CD8+ T cells and can respond to antigen. Those peptides with the ability to induce positive selection were all variants of the antigenic peptide and were identified as TCR antagonist peptides for this receptor. One peptide tested, E1, induced positive selection on the beta 2M(-/-) background but negative selection on the beta 2M(+/-) background. These results show that the process of positive selection is exquisitely peptide specific and sensitive to extremely low ligand density and support the notion that low efficacy ligands mediate positive selection.

Amino Acid Sequence↗

PubMed1997

Positive selection and transplantation of peripheral CD34+ progenitor cells: feasibility and purging efficacy in pediatric patients with neuroblastoma.

Peripheral stem cells were mobilized and collected in 20 children with stage 4 neuroblastoma. A total of 37 leukaphereses were performed in the 20 patients. The mean number of collected cells was 5.6 +/- 2.4 x 10(8)/kg (range 1.9-10.5), and the number of collected CD34+ progenitors was 6.1 +/- 6.3 x 10(6)/kg (range 0.75-21.7). CD34-positive selection was performed using the CellPro method. Of the adsorbed cells, 42 +/- 20% (range 4.3-76.6) stained positively for CD34, and the number of positively selected CD34+ cells was 2.0 +/- 1.9 x 10(6)/kg (range 0.09-7.1). The mean recovery of CD34+ cells was 36 +/- 20% (range 6-67). For detection of contaminating neuroblastoma cells before and after CD34-positive selection, a murine antidisialoganglioside GD2 antibody (14.G2a) was used, followed by the alkaline phosphatase antialkaline phosphatase (APAAP) method. Before the positive selection, various numbers of contaminating neuroblastoma cells were found in the leukaphereses of 7 patients. After positive selection, neuroblastoma cells were still detectable in all 7 patients, with a mean log depletion of tumor cells of 1.41 +/- 0.45 (range 0.69-2.13). In 1 patient, contaminating neuroblastoma cells were found only after CD34-positive selection. In 15 of the 20 patients, high-dose chemotherapy was performed, and positively selected CD34+ cells were reinfused in 12 patients. In 10 of these, the mean time to reach > 0.5 x 10(9)/L granulocytes was 12.3 +/- 1.7 days (range 10-16). One patient died at day 7 due to sepsis, and in 1 patient the backup was given at day 15. Because of the low number of collected CD34+ cells, 3 patients were grafted with a combination of unmanipulated PBSC and CD34+ progenitors. In summary, we have shown that positive selection of peripheral CD34+ progenitors is feasible in pediatric patients. However, strategies to improve the recovery of the CD34+ cells and the purging efficacy of this method (i.e., higher enrichment of CD34+ cells, combination of positive and negative selection methods) should be evaluated further.

Animals↗

PubMed2002

Increased positive selection pressure in persistent (SSPE) versus acute measles virus infections.

We compared the extent of positive selection acting on acute and persistent strains of measles virus (MV). Far stronger positive selection was found in the fusion (F) and haemagglutinin (H) genes from subacute sclerosing panencephalitis (SSPE) compared to acute MV cases. Most of the positively selected sites identified in these surface glycoprotein genes from SSPE cases correspond to structural, functional or antigenic areas, and could not be explained by the effects of cell passaging. The correlations between selected sites and functional studies of MV are discussed in detail with reference to the maintenance of persistent infection. No positive selection was found in the matrix (M) gene from acute cases of MV and the effects of including hypermutated SSPE M gene sequences in phylogenetic inference were also explored. Finally, using H gene data, we estimated the rate of molecular evolution for SSPE strains as 3.4 x 10(-4) substitutions/site/year, which is similar to previous estimates obtained for acute strains.

Evolution, Molecular↗

PubMed2000

Competition for specific intrathymic ligands limits positive selection in a TCR transgenic model of CD4+ T cell development.

Efficient positive selection of a broad repertoire of T cells is dependent on the presentation of a diverse array of endogenous peptides on MHC molecules in the thymus. It is unclear, however, whether the development of individual TCR specificities is influenced by the abundance of their selecting ligands. To examine this, we analyzed positive selection in a transgenic mouse carrying a TCR specific for the human CLIP:I-Ab class II complex. We found that these mice exhibit significantly reduced CD4+ T cell development compared with two other transgenic mice carrying TCRs selected on I-Ab. Moreover, many of the selected cells in these mice express endogenous and transgenic receptors as a consequence of dual TCRalpha expression. Dramatic enhancement of the selection efficiency is observed, however, when fewer transgenic cells populate the thymus in mixed bone marrow chimeras. These results suggest that positive selection is limited by the availability of selecting peptides in the thymus. This becomes apparent when large numbers of thymocytes compete for such peptides in TCR transgenic animals. Under such conditions, thymocytes appear to undergo further TCRalpha gene rearrangement to produce a receptor that may be selected more efficiently by other thymic self-peptides.

Animals↗

PubMed1994

The ligand for positive selection of T lymphocytes in the thymus.

T cells are spared from programmed cell death in the thymus after an appropriate interaction between the T-cell receptor and a self peptide/MHC complex; this step is referred to as positive selection. Recent work has focused on precise identification of the positively selecting ligand, and the cell that presents it. First, it was shown that bone marrow derived cells or fibroblasts can substitute for epithelial cells in providing the ligand for positive selection. Second, in a T-cell receptor transgenic system, variants of the antigenic peptides were found to induce positive selection. Peptides that served as antagonists or weak agonists for the T-cell receptor efficiently selected immature thymocytes for survival. It appears that the peptide ligands for positive selection of T cells are self peptides, which serve as mimics or look alikes for the universe of pathogen peptides. The challenge remains to identify a naturally occurring thymic self peptide that can cause positive selection and determine the range of reactivities to foreign peptides which it can select.

Animals↗

PubMed1999

Specialized ability of thymic epithelial cells to mediate positive selection does not require expression of the steroidogenic enzyme p450scc.

Thymic epithelial cells are uniquely efficient in mediating positive selection, suggesting that in addition to providing peptide/MHC complexes for TCR ligation, they may also provide additional support for this process. Recent studies have shown that although engagement of either the TCR or glucocorticoid (GC) receptors can individually induce apoptosis in thymocytes, together these signals are mutually antagonistic. This had led to the suggestion that local GC production by thymic epithelial cells, by opposing TCR signaling for apoptosis, provides the basis of the ability of these cells to mediate thymocyte positive selection. In this paper we have examined this possibility directly and shown that highly purified cortical epithelial cells, which have the functional ability to mediate positive selection in reaggregate cultures, do not express mRNA for the key steroidogenic enzyme P405scc. Thus we conclude that the ability of thymic epithelial cells to support positive selection does not rely on their ability to produce GC. However, we find that P450scc mRNA is up-regulated in thymocytes on the initiation of positive selection, raising the possibility that any local protective effect of steroid production is mediated at the level of thymocytes themselves.

Animals↗

PubMed1994

A positively selecting thymic epithelial cell line lacks costimulatory activity.

The participation of costimulatory molecule interactions in positive selection of T lymphocytes was addressed by assessing the ability of a positively selecting thymic epithelial cell (TEC) line, 427.1, to stimulate allospecific CTL responses. Stimulation of H-2s spleen cells with the H-2b expressing 427.1 line does not result in the generation of cells capable of lysing H-2b target cell lines. The level of expression of MHC class I molecules by 427.1 is lower than that found in other stimulatory TEC lines. However, this finding does not account for the nonstimulatory phenotype. Up-regulation of MHC class I did not result in stimulation, and fusion of 427.1 cells with stimulatory TEC resulted in a line with low MHC class I molecule expression and stimulatory phenotype. The TEC line 427.1 does not express the costimulatory molecule B7/BB1, and transfection of the B7/BB1-encoding DNA results in expression of the molecule and conversion into a stimulatory phenotype, demonstrating directly that the non-stimulatory phenotype is a result of lack of costimulation. However, B7/BB1 expression does not improve the ability of 427.1 TECs to induce positive selection. Intrathymic injection of the B7/BB1 transfected, compared with mock transfected 427.1 cells, rescued fewer CD8+ mature thymocytes in beta 2-microglobulin negative mice. Therefore, unlike the peripheral T cell responses to Ag, positive selection of T cells in the thymus may not depend on the costimulation provided by the presenting cell.

Animals↗

PubMed1995

Distinct differentiative stages of CD4+CD8+ thymocyte development defined by the lack of coreceptor binding in positive selection.

Cortical CD4+CD8+ thymocytes mature into CD4+ or CD8+ thymocytes through a process termed positive selection. To better define differentiative stages of CD4+CD8+ thymocyte development in positive selection, we performed a phenotypic analysis of CD4+CD8+ thymocytes from H-Y mice mated to various genetic backgrounds. We have previously shown that coordinate binding of the H-Y TCR and the CD8 coreceptor to the restricting Db MHC class I molecule is required for the efficient positive selection of this TCR. In this study we have used TCR, CD5, and CD45 expression levels as markers for thymocyte maturation. Lack of CD8/Db interaction was achieved by introducing a mutation that abrogates CD8 binding in the alpha 3 domain of Db. We found that the absence of coreceptor ligation prevented TCR up-regulation in CD4+CD8+ thymocytes and resulted in a developmental arrest characterized by low levels of TCR and CD45. We have previously shown that deletion of CD4+CD8+ thymocytes expressing the H-Y TCR is facilitated by CD8 coreceptor ligation. Here we show that expression of the deleting ligand in the absence of coreceptor ligation caused CD5 up-regulation without concomitant TCR or CD45 up-regulation in CD4+CD8+ thymocytes. In a beta 2-microglobulin null background, introduction of the H-Y TCR caused the majority of CD4+CD8+ thymocytes to express an unusually low level of of the CD5 activation marker, suggesting that a low-affinity or noncognate TCR/MHC interaction may be required for initial CD5 up-regulation to intermediate levels. Collectively, these observations favor a maturational process in positive selection in which CD5 up-regulation precedes CD45 and TCR up-regulation.

Animals↗

PubMed2002

Immune-mediated positive selection drives human immunodeficiency virus type 1 molecular variation and predicts disease duration.

Using likelihood-based evolutionary methods, we demonstrate that the broad genetic diversity of human immunodeficiency virus type 1 (HIV-1) in an infected individual is a consequence of site-specific positive selection for diversity, a likely consequence of immune recognition. In particular, the extent of positive selection appears to be a good predictor of disease duration. Positively selected sites along HIV-1 partial env sequences are numerous but not distributed uniformly. In a sample of eight patients studied longitudinally, the proportion of sites per sample under positive selection was a statistically significant predictor of disease duration. Among long-term progressors, positive selection persisted at sites over time and appears to be associated with helper T-cell epitopes. In contrast, sites under positive selection shifted from one longitudinal sample to the next in short-term progressors. Our study is consistent with the hypothesis that a broad and persistent immunologic response is associated with a slower rate of disease progression. In contrast, narrow, shifting immune responses characterize short-term progressors.

Disease Progression↗

PubMed2003

Positive selection within sperm-egg adhesion domains of fertilin: an ADAM gene with a potential role in fertilization.

Genes with a role in fertilization show a common pattern of rapid evolution. The role played by positive selection versus lack of selective constraints has been more difficult to establish. One problem arises from attempts to detect selection in an overall gene sequence analysis. I have analyzed the pattern of molecular evolution of fertilin, a gene coding for a heterodimeric sperm protein belonging to the ADAM (A disintegrin and A metalloprotease) gene family. A nonsynonymous to synonymous rate ratio (d(N)/d(S)) analysis for different protein domains of fertilin alpha and fertilin beta showed d(N)/d(S) < 1, suggesting that purifying selection has shaped fertilin's evolution. However, an analysis of the distribution of single positively selected codon sites using phylogentic analysis by maximum likelihood (PAML) showed sites within adhesion domains (disintegrin and cysteine-rich) of fertilin beta evolving under positive selection. The region 3' to the EGF-like domain of fertilin alpha, where the transmembrane and cytoplasmic tail regions are supposed to be localized, showed higher d(N) and d(S) than any other fertilin alpha region. However, it was not possible to identify positively selected codon sites due to ambiguous alignments of the carboxy-end region (ClustalX vs. DiAlign2). When this region was excluded from the PAML analysis, most single positively selected codon sites were concentrated within adhesion domains (cysteine-rich and EGF-like). The use of an ancestral sequence prior to a recent duplication event of fertilin alpha among non-Hominidae primates (Macaca, Papio, and Saguinus) revealed that the duplication is partially responsible for masking the detection of positively selected sites within the disintegrin domain. Finally, most ADAM genes with a potential role in sperm maturation and/or fertilization showed significantly higher d(N) estimates than other ADAM genes.

ADAM Proteins↗

PubMed1999

Class I MHC molecules on hematopoietic cells can support intrathymic positive selection of T cell receptor transgenic T cells.

The identity of cells that mediate positive selection of CD8(+) T cells was investigated in two T cell receptor (TCR) transgenic systems. Irradiated beta(2)-microglobulin mutant mice or mice with mutations in both the K(b) and D(b) genes were repopulated with fetal liver cells from class I(+) TCR transgenic mice. In the case of the 2C TCR, mature transgene-expressing CD8(+) T cells appeared in the thymuses of the chimeras and in larger numbers in the peripheral lymphoid organs. These CD8(+) T cells were functional, exhibited a naive, resting phenotype, and were mostly thymus-dependent. Their development depended on donor cell class I expression. These results establish that thymic hematopoietic cells can direct positive selection of CD8(+) T cells expressing a conventional TCR. In contrast, no significant development of HY (male antigen)-TCR(+) CD8(+) T cells was observed in class I(+) into class I-deficient chimeras. These data suggest that successful positive selection directed by hematopoietic cells depends on specific properties of the TCR or its thymic ligands. The possibility that hematopoietic cell-induced, positive selection occurs only with TCRs that exhibit relatively high avidity interactions with selecting ligands in the thymus is discussed.

Animals↗

PubMed2001

Elevated Bcl-2 is not a causal event in the positive selection of T cells.

T cell development is characterized by the induction of apoptosis in most immature thymocytes and the rescue from apoptosis of a small proportion of cells by the process of positive selection.Up-regulation of the anti-apoptotic molecule Bcl-2 is associated with thymocytes undergoing positive selection and a bcl-2 transgene promotes the generation of mature T cells. In contrast,mice transgenic for the pro-apoptotic molecule Bax show impaired T cell maturation. We have used fetal thymic organ culture to determine the action of Bcl-2 and Bax on positive selection of thymocytes. Our data show that Bcl-2 and Bax do not alter the number of thymocytes positively selected by a defined peptide ligand. This implies that Bcl-2 and Bax alter the production of mature T cells in vivo by influencing thymocyte viability rather than by direct action on positive selection. It also presents a solution to the 'chicken-and-egg' scenario relating to Bcl-2 up-regulation and positive selection. The data suggest that the up-regulation of Bcl-2 associated with T cell maturation is a consequence of positive selection rather than a cause of it.

Animals↗

PubMed1995

New ccdB positive-selection cloning vectors with kanamycin or chloramphenicol selectable markers.

Recently, we described pKIL18/19 positive-selection cloning vectors which rely on the inactivation of the cytotoxic ccdB gene [Bernard et al., Gene 148 (1994) 71-74]. They are new tools for simplifying gene cloning/sequencing procedures, as only recombinant DNA allow the formation of viable colonies. To enhance positive-selection capabilities, putative translational reinitiation codons located within the pKIL18/19 ccdB were modified by site-directed mutagenesis. New pKIL-derived vectors with kanamycin (KmR) or chloramphenicol (CmR) resistance-encoding genes were also constructed. In addition, a new host derived from the DH2 strain was developed.

Bacterial Proteins↗