An adult thymic stromal-cell suspension model for in vitro positive selection.

Presented here is a cell-suspension model for positive selection using thymocytes from alphabeta-TCR (H-2Db-restricted) transgenic mice specific to the lymphocytic choriomeningitis virus (LCMV) on a nonselecting MHC background (H-2d or TAP-1 -/-), cocultured with freshly isolated adult thymus stromal cells of the selecting MHC type. The thymic stromal cells alone induced positive selection of functional CD4- CD8+ cells whose kinetics and efficiency were enhanced by nominal peptide. Fibroblasts expressing the selecting MHC alone did not induce positive selection; however, together with nonselecting stroma and nominal peptide, there was inefficient positive. These results suggest multiple signaling in positive selection with selection events able to occur on multiple-cell types. The ease with which this model can be manipulated should greatly facilitate the resolution of the mechanisms of positive selection in normal and pathological states.

Comparative genomics of chemosensory protein genes reveals rapid evolution and positive selection in ant-specific duplicates.

Gene duplications can have a major role in adaptation, and gene families underlying chemosensation are particularly interesting due to their essential role in chemical recognition of mates, predators and food resources. Social insects add yet another dimension to the study of chemosensory genomics, as the key components of their social life rely on chemical communication. Still, chemosensory gene families are little studied in social insects. Here we annotated chemosensory protein (CSP) genes from seven ant genomes and studied their evolution. The number of functional CSP genes ranges from 11 to 21 depending on species, and the estimated rates of gene birth and death indicate high turnover of genes. Ant CSP genes include seven conservative orthologous groups present in all the ants, and a group of genes that has expanded independently in different ant lineages. Interestingly, the expanded group of genes has a differing mode of evolution from the orthologous groups. The expanded group shows rapid evolution as indicated by a high dN/dS (nonsynonymous to synonymous changes) ratio, several sites under positive selection and many pseudogenes, whereas the genes in the seven orthologous groups evolve slowly under purifying selection and include only one pseudogene. These results show that adaptive changes have played a role in ant CSP evolution. The expanded group of ant-specific genes is phylogenetically close to a conservative orthologous group CSP7, which includes genes known to be involved in ant nestmate recognition, raising an interesting possibility that the expanded CSPs function in ant chemical communication.

TCR-alpha CDR3 loop audition regulates positive selection.

How positive selection molds the T cell repertoire has been difficult to examine. In this study, we use TCR-beta-transgenic mice in which MHC shapes TCR-alpha use. Differential AV segment use is directly related to the constraints placed on the composition of the CDR3 loops. Where these constraints are low, efficient selection of alphabeta pairs follows. This mode of selection preferentially uses favored AV-AJ rearrangements and promotes diversity. Increased constraint on the alpha CDR3 loops leads to inefficient selection associated with uncommon recombination events and limited diversity. Further, the two modes of selection favor alternate sets of AJ segments. We discuss the relevance of these findings to the imprint of self-MHC restriction and peripheral T cell activation.

Patterns of positive selection in seven ant genomes.

The evolution of ants is marked by remarkable adaptations that allowed the development of very complex social systems. To identify how ant-specific adaptations are associated with patterns of molecular evolution, we searched for signs of positive selection on amino-acid changes in proteins. We identified 24 functional categories of genes which were enriched for positively selected genes in the ant lineage. We also reanalyzed genome-wide data sets in bees and flies with the same methodology to check whether positive selection was specific to ants or also present in other insects. Notably, genes implicated in immunity were enriched for positively selected genes in the three lineages, ruling out the hypothesis that the evolution of hygienic behaviors in social insects caused a major relaxation of selective pressure on immune genes. Our scan also indicated that genes implicated in neurogenesis and olfaction started to undergo increased positive selection before the evolution of sociality in Hymenoptera. Finally, the comparison between these three lineages allowed us to pinpoint molecular evolution patterns that were specific to the ant lineage. In particular, there was ant-specific recurrent positive selection on genes with mitochondrial functions, suggesting that mitochondrial activity was improved during the evolution of this lineage. This might have been an important step toward the evolution of extreme lifespan that is a hallmark of ants.

Promoter IV of the class II transactivator gene is essential for positive selection of CD4+ T cells.

Major histocompatibility complex class II (MHCII) expression is regulated by the transcriptional coactivator CIITA. Positive selection of CD4(+) T cells is abrogated in mice lacking one of the promoters (pIV) of the Mhc2ta gene. This is entirely due to the absence of MHCII expression in thymic epithelia, as demonstrated by bone marrow transfer experiments between wild-type and pIV(-/-) mice. Medullary thymic epithelial cells (mTECs) are also MHCII(-) in pIV(-/-) mice. Bone marrow-derived, professional antigen-presenting cells (APCs) retain normal MHCII expression in pIV(-/-) mice, including those believed to mediate negative selection in the thymic medulla. Endogenous retroviruses thus retain their ability to sustain negative selection of the residual CD4(+) thymocytes in pIV(-/-) mice. Interestingly, the passive acquisition of MHCII molecules by thymocytes is abrogated in pIV(-/-) mice. This identifies thymic epithelial cells as the source of this passive transfer. In peripheral lymphoid organs, the CD4(+) T-cell population of pIV(-/-) mice is quantitatively and qualitatively comparable to that of MHCII-deficient mice. It comprises a high proportion of CD1-restricted natural killer T cells, which results in a bias of the V beta repertoire of the residual CD4(+) T-cell population. We have also addressed the identity of the signal that sustains pIV expression in cortical epithelia. We found that the Jak/STAT pathways activated by the common gamma chain (CD132) or common beta chain (CDw131) cytokine receptors are not required for MHCII expression in thymic cortical epithelia.

Selectome update: quality control and computational improvements to a database of positive selection.

Selectome (http://selectome.unil.ch/) is a database of positive selection, based on a branch-site likelihood test. This model estimates the number of nonsynonymous substitutions (dN) and synonymous substitutions (dS) to evaluate the variation in selective pressure (dN/dS ratio) over branches and over sites. Since the original release of Selectome, we have benchmarked and implemented a thorough quality control procedure on multiple sequence alignments, aiming to provide minimum false-positive results. We have also improved the computational efficiency of the branch-site test implementation, allowing larger data sets and more frequent updates. Release 6 of Selectome includes all gene trees from Ensembl for Primates and Glires, as well as a large set of vertebrate gene trees. A total of 6810 gene trees have some evidence of positive selection. Finally, the web interface has been improved to be more responsive and to facilitate searches and browsing.

The branch-site test of positive selection is surprisingly robust but lacks power under synonymous substitution saturation and variation in GC

Positive selection is widely estimated from protein coding sequence alignments by the nonsynonymous-to-synonymous ratio omega. Increasingly elaborate codon models are used in a likelihood framework for this estimation. Although there is widespread concern about the robustness of the estimation of the omega ratio, more efforts are needed to estimate this robustness, especially in the context of complex models. Here, we focused on the branch-site codon model. We investigated its robustness on a large set of simulated data. First, we investigated the impact of sequence divergence. We found evidence of underestimation of the synonymous substitution rate for values as small as 0.5, with a slight increase in false positives for the branch-site test. When dS increases further, underestimation of dS is worse, but false positives decrease. Interestingly, the detection of true positives follows a similar distribution, with a maximum for intermediary values of dS. Thus, high dS is more of a concern for a loss of power (false negatives) than for false positives of the test. Second, we investigated the impact of GC content. We showed that there is no significant difference of false positives between high GC (up to similar to 80%) and low GC (similar to 30%) genes. Moreover, neither shifts of GC content on a specific branch nor major shifts in GC along the gene sequence generate many false positives. Our results confirm that the branch-site is a very conservative test.

Optimization strategies for fast detection of positive selection on phylogenetic trees.

MOTIVATION: The detection of positive selection is widely used to study gene and genome evolution, but its application remains limited by the high computational cost of existing implementations. We present a series of computational optimizations for more efficient estimation of the likelihood function on large-scale phylogenetic problems. We illustrate our approach using the branch-site model of codon evolution. RESULTS: We introduce novel optimization techniques that substantially outperform both CodeML from the PAML package and our previously optimized sequential version SlimCodeML. These techniques can also be applied to other likelihood-based phylogeny software. Our implementation scales well for large numbers of codons and/or species. It can therefore analyse substantially larger datasets than CodeML. We evaluated FastCodeML on different platforms and measured average sequential speedups of FastCodeML (single-threaded) versus CodeML of up to 5.8, average speedups of FastCodeML (multi-threaded) versus CodeML on a single node (shared memory) of up to 36.9 for 12 CPU cores, and average speedups of the distributed FastCodeML versus CodeML of up to 170.9 on eight nodes (96 CPU cores in total).Availability and implementation: ftp://ftp.vital-it.ch/tools/FastCodeML/. CONTACT: selectome@unil.ch or nicolas.salamin@unil.ch.

Classes Alléliques d’Haplotypes et Sélection Positive dans le Génome Humain

L'identification de régions génomiques cibles de la sélection naturelle positive permet de mieux comprendre notre passé évolutif et de trouver des variants génétiques fonctionnels importants. Puisque la fréquence des allèles sélectionnés augmente dans la population, la sélection laisse des traces sur les séquences d'ADN et ces empreintes sont détectées lorsque la variabilité génétique d'une région est différente de celle attendue sous neutralité sélective. On propose une nouvelle approche pour analyser les données de polymorphismes : le calcul des classes alléliques d’haplotypes (HAC), permettant d'évaluer la diversité globale des haplotypes en étudiant leur composition allélique. L'idée de l'approche est de déterminer si un site est sous sélection positive récente en comparant les distributions des HAC obtenues pour les deux allèles de ce site. Grâce à l'utilisation de données simulées, nous avons étudié ces distributions sous neutralité et sous sélection en testant l'effet de différents paramètres populationnels. Pour tester notre approche empiriquement, nous avons analysé la variation génétique au niveau du gène de lactase dans les trois populations inclues dans le projet HapMap.

Positive selection results in frequent reversible amino acid replacements in the G protein gene of human respiratory syncytial virus

Human respiratory syncytial virus (HRSV) is the major cause of lower respiratory tract infections in children under 5 years of age and the elderly, causing annual disease outbreaks during the fall and winter. Multiple lineages of the HRSVA and HRSVB serotypes co-circulate within a single outbreak and display a strongly temporal pattern of genetic variation, with a replacement of dominant genotypes occurring during consecutive years. In the present study we utilized phylogenetic methods to detect and map sites subject to adaptive evolution in the G protein of HRSVA and HRSVB. A total of 29 and 23 amino acid sites were found to be putatively positively selected in HRSVA and HRSVB, respectively. Several of these sites defined genotypes and lineages within genotypes in both groups, and correlated well with epitopes previously described in group A. Remarkably, 18 of these positively selected tended to revert in time to a previous codon state, producing a flipflop phylogenetic pattern. Such frequent evolutionary reversals in HRSV are indicative of a combination of frequent positive selection, reflecting the changing immune status of the human population, and a limited repertoire of functionally viable amino acids at specific amino acid sites.

Expression of human cathepsin L or human cathepsin V in mouse thymus mediates positive selection of T helper cells in cathepsin L knock-out mice

A genetic deficiency of the cysteine protease cathepsin L (Ctsl) in mice results in impaired positive selection of conventional CD4+ T helper cells as a result of an incomplete processing of the MHC class II associated invariant chain or incomplete proteolytic generation of positively selecting peptide ligands. The human genome encodes, in contrast to the mouse genome, for two cathepsin L proteases, namely cathepsin L (CTSL) and cathepsin V (CTSV; alternatively cathepsin L2). In the human thymic cortex, CTSV is the predominately expressed protease as compared to CTSL or other cysteine cathepsins. In order to analyze the functions of CTSL and CTSV in the positive selection of CD4+ T cells we employed Ctsl knock-out mice crossed either with transgenic mice expressing CTSL under the control of its genuine human promoter or with transgenic mice expressing CTSV under the control of the keratin 14 (K14) promoter, which drives expression to the cortical epithelium. Both human proteases are expressed in the thymus of the transgenic mice, and independent expression of both CTSL and CTSV rescues the reduced frequency of CD4+ T cells in Ctsl-deficient mice. Moreover, the expression of the human cathepsins does not change the number of CD4+CD25+Foxp3+ regulatory T cells, but the normalization of the frequency of conventional CD4+ T cell in the transgenic mice results in a rebalancing of conventional T cells and regulatory T cells. We conclude that the functional differences of CTSL and CTSV in vivo are not mainly determined by their inherent biochemical properties, but rather by their tissue specific expression pattern.

Continental-Scale Footprint of Balancing and Positive Selection in a Small Rodent (Microtus arvalis)

Genetic adaptation to different environmental conditions is expected to lead to large differences between populations at selected loci, thus providing a signature of positive selection. Whereas balancing selection can maintain polymorphisms over long evolutionary periods and even geographic scale, thus leads to low levels of divergence between populations at selected loci. However, little is known about the relative importance of these two selective forces in shaping genomic diversity, partly due to difficulties in recognizing balancing selection in species showing low levels of differentiation. Here we address this problem by studying genomic diversity in the European common vole (Microtus arvalis) presenting high levels of differentiation between populations (average FST = 0.31). We studied 3,839 Amplified Fragment Length Polymorphism (AFLP) markers genotyped in 444 individuals from 21 populations distributed across the European continent and hence over different environmental conditions. Our statistical approach to detect markers under selection is based on a Bayesian method specifically developed for AFLP markers, which treats AFLPs as a nearly codominant marker system, and therefore has increased power to detect selection. The high number of screened populations allowed us to detect the signature of balancing selection across a large geographic area. We detected 33 markers potentially under balancing selection, hence strong evidence of stabilizing selection in 21 populations across Europe. However, our analyses identified four-times more markers (138) being under positive selection, and geographical patterns suggest that some of these markers are probably associated with alpine regions, which seem to have environmental conditions that favour adaptation. We conclude that despite favourable conditions in this study for the detection of balancing selection, this evolutionary force seems to play a relatively minor role in shaping the genomic diversity of the common vole, which is more influenced by positive selection and neutral processes like drift and demographic history.

Patterns of positive selection in seven ant genomes

The evolution of ants is marked by remarkable adaptations that allowed the development of very complex social systems. To identify how ant-specific adaptations are associated with patterns of molecular evolution, we searched for signs of positive selection on amino-acid changes in proteins. We identified 24 functional categories of genes which were enriched for positively selected genes in the ant lineage. We also reanalyzed genome-wide datasets in bees and flies with the same methodology, to check whether positive selection was specific to ants or also present in other insects. Notably, genes implicated in immunity were enriched for positively selected genes in the three lineages, ruling out the hypothesis that the evolution of hygienic behaviors in social insects caused a major relaxation of selective pressure on immune genes. Our scan also indicated that genes implicated in neurogenesis and olfaction started to undergo increased positive selection before the evolution of sociality in Hymenoptera. Finally, the comparison between these three lineages allowed us to pinpoint molecular evolution patterns that were specific to the ant lineage. In particular, there was ant-specific recurrent positive selection on genes with mitochondrial functions, suggesting that mitochondrial activity was improved during the evolution of this lineage. This might have been an important step toward the evolution of extreme lifespan that is a hallmark of ants.

T cell positive selection by a high density, low affinity ligand

Interaction of the αβ T cell receptor (TCR) with major histocompatibility (MHC) molecules occupied with any of a large collection of peptides derived from self proteins is a critical step in driving T cell “positive” selection in the thymus. Interaction with this same pool of self-peptide/MHC ligands deletes T cells with potential self-reactivity. To examine how T cells survive both of these processes to form a self-tolerant mature repertoire, mice were constructed whose entire class II MHC IEk specific repertoire was positively selected on a single peptide covalently attached to the IEk molecule. In these mice T cells were identified that could respond to a variant of the positively selecting peptide bound to IEk. The affinities of the TCRs from these T cells for the positively selecting ligand were extremely low and at least 10-fold less than those for the activating ligand. These results support the theory that positive selection is driven by TCR affinities lower than those involved in T cell deletion or activation and that, if present at high concentration, even very low affinity ligands can positively select.

Altered thymic positive selection and intracellular signals in Cbl-deficient mice

Cbl is the product of the protooncogene c-cbl and is involved in T cell antigen receptor (TCR)-mediated signaling. To understand the role of Cbl for immune system development and function, we generated a Cbl-deficient mouse strain. In Cbl-deficient mice, positive selection of the thymocytes expressing major histocompatibility complex class II-restricted transgenic TCR was significantly enhanced. Two factors may have contributed to the altered thymic selection. First, Cbl deficiency markedly up-regulated the activity of ZAP-70 and mitogen-activated protein kinases. The mitogen-activated protein kinase pathway was shown previously to be involved in thymic positive selection. Second, Cbl-deficient thymocytes expressed CD3 and CD4 molecules at higher levels, which consequently may increase the avidity of TCR/major histocompatibility complex/coreceptor interaction. Thus, Cbl plays a novel role in modulating TCR-mediated multiple signaling pathways and fine-tunes the signaling threshold for thymic selection.