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PubMed1992

A cell line that can induce thymocyte positive selection.

The thymus positively selects thymocytes that bear T-cell receptors which recognize antigen presented by self major histocompatibility complex (MHC) proteins. Positive selection is usually driven by MHC products on radiation-resistant cortical epithelial cells. It is unknown whether positive selection is mediated by all thymic epithelial cells or by some specialized subsets. Here we introduce an H-2b-expressing thymic epithelial cell line into the thymuses of lethally irradiated H-2k animals reconstituted with H-2b/k F1 BM or fetal liver cells. I-Ab-restricted T cells are found in these animals, demonstrating that selection occurs on the introduced epithelial cells.

Animals↗
PubMed2000

Fetal nucleated erythrocyte recovery: fluorescence activated cell sorting-based positive selection using anti-gamma globin versus magnetic activated cell sorting using anti-CD45 depletion and anti-gamma globin positive selection.

BACKGROUND: Fluorescence activated cell sorting (FACS)-based anti-gamma (gamma) positive selection and magnetic activated cell sorting (MACS)-based anti-CD45 depletion followed by anti-gamma positive staining have been two of the most frequently used methods to isolate fetal cells from maternal blood. To date, there has been no direct comparison of fetal cell recovery by these two methods. This study was designed to address this issue. METHODS: Fluorescence in situ hybridization (FISH) was performed on nucleated anti-gamma positive cells using X and Y probes. Twenty-four maternal blood samples were obtained immediately after elective termination of pregnancy to ensure a detectable number of fetal cells. RESULTS: The yield and purity of fetal nucleated erythrocytes (FNRBCs) was statistically higher in FACS sorted samples (P < 0.01). The specificity of staining for FNRBCs was statistically higher in MACS sorted samples (P < 0.01). CONCLUSIONS: The data from this study demonstrate that both techniques have benefits and limitations. FACS has the advantage of having higher yield, higher purity, higher FISH efficiency and ease in microscope analysis, and MACS has the advantage of having higher specificity and less cell loss during FISH.

Antigens, CD↗
PubMed1996

Positive selection, cloning vectors for gram-positive bacteria based on a restriction endonuclease cassette.

Lactococcus lactis contains numerous restriction and modification (R/M) systems of different specificities. A novel IIS type R/M system encoded by the LlaI operon has previously been characterized from the L. lactis conjugative plasmid pTR2030. The LlaI operon is composed of six genes: First, a small regulatory gene llaIC precedes the methylase gene llaIM. The following three genes, llaI.1, llaI.2, llaI.3, are all essential for restriction endonuclease activity and are designed as the restriction cassette llaIR. The forth open reading frame of unknown function follows the llaIR gene cassette. We have successfully subcloned the three llaIR genes, llaI.1, llaI.2, and llaI.3, without llaIM, as a suicide cassette into the three shuttle vectors pTRKL2, pTRKH2, and pBV5030. A promoter (P6) from Lactobacillus acidophilus ATCC4356, which is functional in E. coli, lactococci, and lactobacilli (Djordjevic and Topisirovic, unpublished) was cloned upstream of the three gene cassette. Restriction activity was evaluated in Escherichia coli and several gram-positive bacteria. The llaIR restriction cassette was not functional in E. coli, but its presence was lethal to L. lactis, Lactobacillus gasseri, Lactobacillus plantarum, Lactobacillus johnsonii, Lactobacillus acidophilus, Carnobacterium pisicola, Enterococcus faecalis, Bacillus subtilis, and Leuconostoc gelidum. Several novel, positive selection cloning vectors were developed that can exploit unique cloning sites within the llaIR cassette. Insertions in llaI.1 resulted in complete inactivation of restriction activity and provided unconditional selection for recombinant plasmids in surviving transformants. These positive selection cloning vectors are the first for gram-positive bacteria that are based on a restriction endonuclease cassette. Functional activity of the llaIR genes in various gram-positive bacteria would also enable use of these cloning vectors for positive selection of promoters, terminators, and regulatory sequences across these genera.

Cloning, Molecular↗
PubMed2000

Molecular basis for functional maturation of thymocytes: increase in c-fos translation with positive selection.

In the process of positive selection, immature CD4+8+ double positive (DP) thymocytes expressing TCR reactive to self-MHC by appropriate avidity develop into mature thymocytes. Positive selection involves not only down-regulation of either CD4 or CD8 but also acquisition of immunocompetent potential such as cell proliferation and cytokine production. To understand the molecular basis for such functional maturation during the positive selection process, we examined whether nonselected DP, selected DP, and CD4+8- single positive thymocytes possess the activation potential for signaling pathways from mitogen-activated protein kinases (extracellular signal-regulated kinase and c-Jun N-terminal kinase) to AP-1. In response to stimulation, a marked induction of c-Fos protein expression as well as cell proliferation is detected only in CD4+8- single positive cells but not in selected and nonselected DP cells, though mitogen-activated protein kinase activities and c-fos transcripts are equally induced. In the presence of proteasome inhibitors, c-Fos protein became detectable in selected DP cells but still not in nonselected DP cells, suggesting that DP cells receiving positive selection signals acquire the capacity to translate the c-fos gene, but it may not be sufficiently high to overcome the degradation of c-Fos protein. These data indicate that the translating ability of the c-fos gene is up-regulated in the thymic positive selection process, from nonselected DP to CD4+8- single positive cells through positively selected DP cells. The distinguished responsiveness to stimulation in thymocytes with and without positive selection may be a result in part of the distinct regulation of the c-fos gene at the translational level.

Animals↗
PubMed1999

Differential MHC expression requirements for positive selection of separate TCR Vb families.

Positive selection has been proposed to be involved in protection from diabetes. We examined positive selection by fluorescence-activated cell sorter analyses in thymocytes of protected and susceptible E-transgenic and non-transgenic NOD mice. Three Vb families showed positive selection in E-transgenic mice. Vb6(+)CD4(+) and Vb10(+)CD4(+) thymocytes were found at higher frequencies in both protected NOD-Ea and susceptible NOD-DY mice. The increased frequencies of Vb13(+)CD8(+) thymocytes were found in protected NOD-Ea mice only, and not in susceptible NOD-DY transgenic mice. These three Vb families were further examined in bone-marrow chimeras between NOD-Ea and non-transgenic NOD mice, where we could examine the contribution of E-expressing bone-marrow-derived cells in positive selection. We find that NOD-Ea-->NOD-Ea chimeras have an increased positive selection of Vb13(+)CD8(+) cells and that positive selection is more efficient when both thymic epithelium and bone-marrow-derived cells express the E molecule. This was also seen for Vb6(+)CD4(+) cells. However, for Vb6, bone-marrow-derived cells alone were also capable of positive selection. Positive selection of Vb10(+)CD4(+) cells was restricted to E-expressing thymic epithelium only. For Vb13(+)CD8(+ )cells, we found that positive selection is most efficient with E-expression on both thymic epithelium and bone-marrow-derived cells, although positive selection also occurs with E-positive epithelium only. For Vb6(+) CD4(+) cells, the dominating selecting cells are bone-marrow-derived cells, and Vb10(+)CD4(+ )cells seem to be selected exclusively by the thymic epithelium. Thus, the conditions for positive selection seem to vary considerably between different Vb families.

Animals↗
PubMed1994

In favor of the selective model of positive selection.

The mechanisms of thymocyte commitment towards the CD4+ and CD8+ lineage remain unresolved. Two models--one based on instruction, the other on selection--have previously been proposed. The instructional model has been popularly received based on results of earlier studies. However, our data from MHC class II, class I, and double-deficient mice suggest otherwise. There exists a significant population of CD4+ cells that is intermediate in maturity between CD4+ CD8+ and fully mature CD4+ CD8- thymocytes in class II-deficient animals; an analogous population of CD4-CD8+ cells exists in class I-negative mice. We suggest that a selective model in which two TCR-MHC molecule engagements are required: the first induces a random down-modulation of either CD4 or CD8 and some differentiation; the second, involving the participation of the appropriate coreceptor, permits end-stage differentiation.

Animals↗
PubMed2001

An incremental increase in the complexity of peptides bound to class II MHC changes the diversity of positively selected alpha beta TCRs.

Positive selection of the normal repertoire of TCRs results from low-avidity interactions with a set of self-peptides bound to the MHC molecules expressed by thymic epithelial cells. The contribution of the individual peptide to positive selection remains a matter of debate. Here, for the first time, we show that two covalent class II MHC-peptide complexes positively select different TCRs expressing a common transgenic TCRbeta-chain and endogenous TCRalpha-chains. Simultaneous expression of both A(b)-peptide complexes changed the diversity of positively selected TCRs, indicating an additive and possibly synergistic effect of various peptides in this process.

Animals↗
PubMed2000

Evidence for migration of donor bone marrow stromal cells into recipient thymus after bone marrow transplantation plus bone grafts: A role of stromal cells in positive selection.

Intrathymic T-cell differentiation is characterized by two selection events: positive and negative selection. It has been shown that thymic epithelial cells in the cortex are involved in the positive selection, while macrophages and dendritic cells, derived from hemopoietic stem cells, are involved in the negative selection. Here we investigate whether donor-derived bone marrow stromal cells can migrate into the thymus and participate there in positive selection after bone marrow transplantation plus bone grafts (to recruit bone marrow stromal cells). Allogeneic bone marrow transplantation with or without bone grafts was carried out in the [C57BL/6-->C3H] combination. Fluorescence-activated cell sorter analyses of recipient thymic adherent cells showed that donor-type bone marrow stromal cells exist in the thymus of mice that received bone marrow plus bone grafts but not in the mice that received bone marrow cells alone. Histological examination using confocal microscopy also confirmed the existence of donor-type stromal cells in the thymus of mice that received bone marrow cells plus bones. Both T-cell proliferation and plaque-forming cell assays indicated that the T cells of such mice show donor-type major histocompatibility complex-restriction. These findings strongly suggest that stromal cells can migrate from the bone marrow to the thymus, where they participate in the positive selection of thymocytes.

Animals↗
PubMed1992

Tyrosine kinase triggering in thymocytes undergoing positive selection.

Developing T cells undergo distinct selection processes that determine the T cell receptor (TcR) repertoire. One of these processes is positive selection. Positive selection involves the differentiation to mature T cells of thymocytes bearing TcR capable of recognizing antigens in the context of self-major histocompatibility complex (MHC) molecules. To study the potential involvement of tyrosine phosphorylation in the mechanism of positive selection, we have analyzed the activities of the tyrosine kinases pp56lck and pp59fyn in thymocytes expressing a unique TcR specific for HY antigen+ H-2 Db. Thymocytes undergoing positive selection displayed higher kinase specific activities of pp56lck and pp59fyn than nonselecting thymocytes. Furthermore, these increases in kinase activities were found only in the CD4+CD8+ subpopulation of thymocytes, where the selection process is believed to occur. These data suggest that tyrosine phosphorylation is part of the intracellular signals involved in MHC class I-driven positive selection from CD4+CD8+ immature thymocytes to CD4-CD8+ mature cells.

Animals↗
PubMed1996

Phorbol ester and calcium ionophore can replace TCR signals that induce positive selection of CD4 T cells.

Positive selection of immature thymocytes is a developmental process in which TCR ligation by low avidity interaction induces the generation of mature T cells. However, biochemical signals that can induce positive selection have been unclear. By using TCR-alpha beta- mutant thymus cultures, the present study shows that direct stimulation of intracellular signals by PMA and calcium ionophore ionomycin can induce the generation of mature CD4+8- T cells, bypassing TCR-induced positive selection signals. Interestingly, the concentrations of phorbol ester that induced positive selection were more restricted than those that induced mature T cell activation. Moreover, the combination of phorbol ester and ionomycin restored the generation of CD4+8- T cells in class II MHC- thymus cultures, but did not induce the generation of CD4-8+ T cells in class I MHC- thymus cultures. These results identify that the combination of protein kinase C activation and calcium elevation is the biochemical signal that can induce positive selection of CD4+ T cells.

Animals↗
PubMed1999

Functional similarity and differences between selection-independent CD4-CD8- alphabeta T cells and positively selected CD8 T cells expressing the same TCR and the induction of anergy in CD4-CD8- alphabeta T cells in antigen-expressing mice.

In TCR-alphabeta transgenic mice, CD4-CD8- TCR-alphabeta+ (alphabeta DN) cells arise in the absence of positively selecting MHC molecules and are resistant to clonal deletion in Ag-expressing mice. In this study the activation requirements and functional properties of alphabeta double-negative (DN) cells were compared with those of positively selected CD8+ cells expressing equivalent levels of the same MHC class I-restricted transgenic TCR. We found that positively selected CD8+ cells required a lower density of the antigenic ligand for optimal proliferative responses compared with alphabeta DN cells derived from nonpositively selecting mice. However, when the CD8 coreceptor on CD8+ cells was blocked with an anti-CD8 mAb, both alphabeta DN and CD8+ cells exhibited the same dose-response curve to the antigenic ligand and the same dependence on CD28/B7 costimulation. Positively selected CD8+ cells also differed from alphabeta DN cells in that they differentiated into more efficient killers and IL-2 producers after Ag stimulation, even after CD8 blockade. However, Ag-activated alphabeta DN and CD8+ cells were equally efficient in producing IFN-gamma, suggesting that this functional property is independent of positive selection. We also found that alphabeta DN cells recovered from the lymph nodes of Ag-expressing mice were functionally anergic. This anergic state was associated with defective proliferation and IL-2 production in response to Ag stimulation. These observations indicate that alphabeta DN cells can be anergized in vivo by physiological levels of the antigenic ligand.

Animals↗
PubMed2002

Positive selection vectors.

This review describes information concerning positive selection vectors on their mechanism, classification, property, and limitation. A total of 72 positive selection vectors collected were discussed. Positive selection vectors can reduce background and directly screen transformants containing cloned DNA fragments. The mechanisms to perform positive selection include insertional inactivation and the replacement of functional genes of the vectors. In general, the former is much more convenient than the latter. The functional genes are controlled either by their promoters or by heterologous promoters introduced. On the basis of the structures, positive selection vectors could be classified into five groups. The positive selection vectors are commonly based on the mechanisms of lethal genes and the sensitivity of compounds. The vectors, with molecular weights ranging from 2.6 to 17.0 kb, have diverse genetic markers and wide host ranges, including Escherichia coli, Bacillus, Streptomyces, lactic acid bacteria, yeasts, and mammalian cells. Although some limitations exist for using some positive selection vectors, they are useful in recombinant DNA experiments.

Biotechnology↗
PubMed1999

Genealogical evidence for positive selection in the nef gene of HIV-1.

The pattern and process of evolution in the nef gene of HIV-1 was analyzed within and among patients. Using a maximum likelihood method that allows for variable intensity of selection pressure among codons, strong positive selection was detected in a hemophiliac patient over 30 mo of infection. By reconstructing the process of allele substitution in this patient using parsimony, the synapomorphic amino acid changes separating each time point were found to have high probabilities of being under positive selection, with selective coefficients of at least 3.6%. Positive selection was also detected among 39 nef sequences from HIV-1 subtype B. In contrast, multiple pairwise comparisons of nonsynonymous and synonymous substitution rates provided no good evidence for positive selection and sliding window analyses failed to detect most positively selected sites. These findings demonstrate that positive selection is an important determinant of nef gene evolution and that genealogy-based methods outperform pairwise methods in the detection of adaptive evolution. Mapping the locations of positively selected sites may also be of use in identifying targets of the immune response and hence aid vaccine design.

Acquired Immunodeficiency Syndrome↗
PubMed2004

Positive selection on the human genome.

Positive selection has undoubtedly played a critical role in the evolution of Homo sapiens. Of the many phenotypic traits that define our species--notably the enormous brain, advanced cognitive abilities, complex vocal organs, bipedalism and opposable thumbs--most (if not all) are likely the product of strong positive selection. Many other aspects of human biology not necessarily related to the 'branding' of our species, such as host-pathogen interactions, reproduction, dietary adaptation and physical appearance, have also been the substrate of varying levels of positive selection. Comparative genetics/genomics studies in recent years have uncovered a growing list of genes that might have experienced positive selection during the evolution of human and/or primates. These genes offer valuable inroads into understanding the biological processes specific to humans, and the evolutionary forces that gave rise to them. Here, we present a comprehensive review of these genes, and their implications for human evolution.

Genome, Human↗
PubMed1997

MHC class II molecules are required for initiation of positive selection but not during terminal differentiation of human CD4 single positive thymocytes.

Positive selection of T cell precursors is an MHC dependent, multistep process by which functionally mature CD4+8- helper and CD4-8+ cytotoxic single positive (SP) T cells are generated from immature CD4+8+ double positive (DP) thymocytes. We investigated the requirement for TCR/MHC class II interactions during different stages of positive selection of human CD4 SP thymocytes. We show that sorted CD69- CD4+8+ DP preselection thymocytes cultured in fetal thymus lobes of normal mice were subject to positive selection and differentiated to CD3(high) CD69+, mature CD8 SP, and CD4 SP cells. When cultured in thymus lobes from MHC class II-deficient mice, these precursors failed to develop into mature CD4 SP T cells, indicating that in the hybrid cultures, murine MHC class II molecules are required for the development of mature human CD4 SP T cells. We have previously identified CD4 SP intermediate thymocytes that have received at least some of the signals involved in positive selection, since these cells are CD69+, CD3/TCR(high), and CD8beta- but that are still phenotypically and functionally immature. Here we demonstrate that in contrast to preselection thymocytes, these CD4 SP intermediate thymocytes can give rise to phenotypically mature and functionally CD4 SP progeny both in normal and in MHC class II-deficient thymus lobes. These results suggest that TCR/MHC interactions are required for the initial stages of positive selection, but are not essential during terminal differentiation to functionally mature CD4 SP T cells.

Animals↗
PubMed1995

Strong agonist ligands for the T cell receptor do not mediate positive selection of functional CD8+ T cells.

Positive selection of functional CD8+ T cells expressing an MHC class I-restricted T cell receptor can be induced in fetal thymus organ culture by class I-binding peptides related to the antigenic peptide ligand. Peptides that act as antagonist or weak agonist/antagonist ligands for mature T cells work efficiently in this regard. In the present study, we have investigated whether low concentrations of the original agonist peptide, or variants that still have a strong agonist activity can also mediate positive selection. The antigenic peptide did not induce positive selection at any concentration tested. A strong agonist variant was capable of stimulating the differentiation of TCRhi CD8+ cells, giving the appearance of phenotypic positive selection. However, these cells lacked biological function, since they could not proliferate in response to antigen. The most efficient positive selection resulted with ligands that did not activate mature T cells or stimulate negative selection.

Animals↗
PubMed1998

A procedure for detecting selection in highly variable viral genomes: evidence of positive selection in antigenic regions of capsid protein VP1 of foot-and-mouth disease virus.

A new procedure is described for the detection of positive selection among sequences of viral proteins from highly variable viruses. The approach is based on the estimation of the rates of nonsynonymous to synonymous (ns/s) mutations to the overall genetic distances amongst the sequences compared. Rates of ns/s substitutions were calculated, and the individual profiles were arranged as a function of the genetic distance observed between the complete sequences. The resulting surfaces allowed identification of protein regions whose rates of ns/s substitutions were consistent with the existence of positive selection. This procedure has been applied to the study of a highly variable antigenic protein, VP1, a protein present in foot-and-mouth disease virus (FMDV). The analysis of groups of VP1 sequences corresponding to FMDV serotypes A, O and C, resulted in the identification of two regions, which contribute to an important antigenic site, where positive selection appears to operate.

Antigenic Variation↗
PubMed1998

Two distinct pathways of positive selection for thymocytes.

Most mouse thymocytes undergoing positive selection are found on one of two pathways; the c-Kit+ and the c-Kit- pathways. Here, we show that c-Kit and interleukin-7 receptor (IL-7R)-mediated signals support positive selection during the transition from the subpopulation that first expresses cell surface T cell receptor (TCR)-the TCRalpha/betaloCD4(int)/CD8(int) (DPint) c-Kit+ cells to TCRalpha/betamedc-Kit+ transitional intermediate cells (the c-Kit+ pathway). Cells that fail positive selection on the c-Kit+ pathway become TCRalpha/betaloc-Kit- (DPhi) blasts that appear to undergo alternative TCRalpha rearrangements. The rare DPhic-Kit- blast cells that thus are salvaged for positive selection by expressing a self-major histocompatibility complex selectable TCRalpha/beta up-regulate IL-7R, but not c-Kit, and are the principal progenitors on the c-Kit- pathway; this c-Kit-IL-7R+ pathway is mainly CD4 lineage committed. Cell division is a feature of the TCRlo-medc-Kit+ transition, but is not essential for CD4 lineage maturation from DPhic-Kit- blasts. In this view, positive selection on the c-Kit- path results from a salvage of cells that failed positive selection on the c-Kit+ path.

Animals↗