B cell subsets, idiotype selection: positive selection for some B lymphocytes?
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Increased p300 expression inhibits glucocorticoid receptor-T-cell receptor antagonism but does not affect thymocyte positive selection.
Positive selection of T cells is postulated to be dependent on the counterinteraction between glucocorticoid receptor (GR)- and T-cell-receptor (TCR)-induced death signals. In this study we used T-cell-specific expression of p300 to investigate whether GR-TCR cross talk between thymocytes was affected. Activation of the p300-transgenic T cells led to enhanced thymocyte proliferation and increased interleukin 2 production. Thymocyte death, induced by TCR engagement, was no longer prevented by dexamethasone in p300-transgenic mice, indicating an absence of GR-TCR cross-inhibition. This was accompanied by a 50% reduction in the number of thymocytes in p300-transgenic mice. However, the CD4/CD8 profile of thymocytes remained unchanged in p300-transgenic mice. There was no effect on positive selection of the bulk thymocytes or thymocytes with transgenic TCR in p300-transgenic mice. In addition, there was no apparent TCR repertoire "hole" in the selected antigens examined. Our results illustrate a critical role of CBP/p300 in thymic GR-TCR counterinteraction yet do not support the involvement of GR-TCR antagonism in thymocyte positive selection.
Amino Acid Sequence↗
An inhibitory Ig superfamily protein expressed by lymphocytes and APCs is also an early marker of thymocyte positive selection.
Positive selection of developing thymocytes is associated with changes in cell function, at least in part caused by alterations in expression of cell surface proteins. Surprisingly, however, few such proteins have been identified. We have analyzed the pattern of gene expression during the early stages of murine thymocyte differentiation. These studies led to identification of a cell surface protein that is a useful marker of positive selection and is a likely regulator of mature lymphocyte and APC function. The protein is a member of the Ig superfamily and contains conserved tyrosine-based signaling motifs. The gene encoding this protein was independently isolated recently and termed B and T lymphocyte attenuator (Btla). We describe in this study anti-BTLA mAbs that demonstrate that the protein is expressed in the bone marrow and thymus on developing B and T cells, respectively. BTLA is also expressed by all mature lymphocytes, splenic macrophages, and mature, but not immature bone marrow-derived dendritic cells. Although mice deficient in BTLA do not show lymphocyte developmental defects, T cells from these animals are hyperresponsive to anti-CD3 Ab stimulation. Conversely, anti-BTLA Ab can inhibit T cell activation. These results implicate BTLA as a negative regulator of the activation and/or function of various hemopoietic cell types.
Alternative Splicing↗
Thymocytes between the beta-selection and positive selection checkpoints are nonresponsive to IL-7 as assessed by STAT-5 phosphorylation.
Interleukin-7 is widely accepted as a major homeostatic factor involved in T cell development. To assess the IL-7 responsiveness of thymocytes involved in selection processes, we used a new sensitive flow cytometry-based assay to detect intracellular phosphorylation of STAT-5 induced by IL-7 in defined mouse thymocyte subsets. Using this method, we found the earliest thymocyte subset (CD4(-)CD8(-)CD25(-)CD44(+)) to contain both IL-7-responsive and nonresponsive cells. Transition through the next stages of development (CD4(-)CD8(-)CD25(+)CD44(+ and -)) was associated with responsiveness of all thymocytes within these populations. Passage of thymocytes through beta-selection resulted in a significant reduction in IL-7 sensitivity. In the next phases of development (TCR(-) and TCR(low)CD69(-)), thymocytes were completely insensitive to the effects of IL-7. STAT-5 phosphorylation in response to IL-7 was again observed, however, in thymocytes involved in the positive selection process (TCR(low)CD69(+) and TCR(intermediate)). As expected, CD4 and CD8 single-positive thymocytes were responsive to IL-7. These findings delineate an IL-7-insensitive population between the beta-selection and positive selection checkpoints encompassing thymocytes predicted to die by neglect due to failure of positive selection. This pattern of sensitivity suggests a two-signal mechanism by which survival of thymocytes at these checkpoints is governed.
Animals↗
The nature of the peptide/MHC ligand involved in positive selection.
Positive and negative selection in the thymus occur when the TCR on an immature thymocyte engages self peptide/MHC complexes present on epithelial cells. The signaling mechanism which dictates the selection outcome is currently a matter of intense investigation. Here we review experiments that defined the peptide ligands for positive selection. A comparison of these to the peptide ligands that induce negative selection (antigenic peptides) sheds some insight into how a thymocyte interprets peptide/MHC interactions leading to life versus death of the cell.
Animals↗
Positive selection.
The use of a new mode of selection-positive selection-has been demonstrated to be successful in a large variety of monocot and dicot species. This selection differs from more traditional modes of selection in which compounds such as antibiotics or herbicides are used to kill nontransformed cells (negative selection). In the case of positive selection, a transformed cell acquires the ability to metabolize a substrate that it previously could not use (or not use efficiently) and thereby grows out of the mass of nontransformed tissue. Positive selection can be of many types from inactive forms of plant growth regulators that are then converted to active forms by the transferred enzyme to alternative carbohydrate sources that are not utilized efficiently by the nontransformed cells that become available upon transformation with an enzyme that allows them to be metabolized. Nontransformed cells either grow slowly in comparison to transformed cells or not at all. Using positive selection, nontransformed cells may die, but, typically, production of phenolic compounds observed with negative selection markers does not occur. In many cases, this effect contributes to higher transformation efficiencies, as these compounds can negatively influence the growth of transformed cells. The use of one form of positive selection-transformation with phosphomannose isomerase followed by selection on mannose containing media-is presented here as an example.
Indicators and Reagents↗
Alcohol selection and position selection in hamsters caged singly and in groups.
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Alcohol Drinking↗
Thymic stromal cells and positive selection.
The intrathymic differentiation events leading to the development and export of mature T cells tolerant to self yet capable of responding to foreign peptide antigen in the context of self-MHC are clearly both dynamic and complex. The changing phenotype of the developing thymocyte as it migrates through and interacts with the heterogeneous thymic microenvironment and the intracellular signalling events associated with such interactions are being extensively studied, yet many aspects remain poorly defined, such as the precise relationship between stromal cells and thymic selection. Positive and negative selection are crucial events in the development of T cells, leading to a diverse yet non-autoreactive immune system. A breakdown in either of these processes could lead to either a reduced T cell repertoire or the escape into the periphery of autoreactive T cells - both clearly having deleterious consequences for the health of the individual. This review aims to summarise the current status of research in thymic positive selection with emphasis on the role of different cell types and peptides.
Animals↗
CD45 enhances positive selection and is expressed at a high level in large, cycling, positively selected CD4+CD8+ thymocytes.
T-cell development is arrested at the CD4+CD8+ (DP; double-positive) stage of thymocyte development in CD45 null mice. However, the mechanism by which CD45 participates in the positive selection of T cells remains to be investigated. In this report we describe a DP thymocyte population that associates positive selection with expression of high levels of CD45, CD4 and CD8. DP thymocytes of this phenotype are large, cycling cells and represent approximately 20% of DP thymocytes in normal mice. In mice expressing a transgenic T-cell receptor (TCR) specific for the male antigen presented by H-2Db (H-Y TCR), the up-regulation of TCR, CD5 and CD69 in this large DP population occurred in a major histocompatibility complex (MHC)-restricted manner. To investigate further the role of CD45 in positive selection, we determined whether thymocytes that expressed a transgenic CD45RO molecule under the control of the proximal lck promoter can influence the positive selection of T cells in H-Y TCR transgenic mice. It was found that in female H-Y TCR transgenic mice, MHC-restricted positive selection of CD4- CD8+ H-Y TCR+ thymocytes was enhanced by increased CD45RO expression. Thus, CD45 increases the efficacy of positive selection of CD4- CD8+ thymocytes that express H-Y TCR.
Animals↗
Programmed death-1 (PD-1):PD-ligand 1 interactions inhibit TCR-mediated positive selection of thymocytes.
Positive selection during thymocyte development is driven by the affinity and avidity of the TCR for MHC-peptide complexes expressed in the thymus. In this study, we show that programmed death-1 (PD-1), a member of the B7/CD28 family of costimulatory receptors, inhibits TCR-mediated positive selection through PD-1 ligand 1 (PD-L1):PD-1 interactions. Transgenic mice that constitutively overexpress PD-1 on CD4+CD8+ thymocytes display defects in positive selection in vivo. Using an in vitro model system, we find that PD-1 is up-regulated following TCR engagement on CD4+CD8+ murine thymocytes. Coligation of TCR and PD-1 on CD4+CD8+ thymocytes with a novel PD-1 agonistic mAb inhibits the activation of ERK and up-regulation of bcl-2, both of which are downstream mediators essential for positive selection. Inhibitory signals through PD-1 can overcome the ability of positive costimulators, such as CD2 and CD28, to facilitate positive selection. Finally, defects in positive selection that result from PD-1 overexpression in thymocytes resolve upon elimination of PD-L1, but not PD-1 ligand 2, expression. PD-L1-deficient mice have increased numbers of CD4+CD8+ and CD4+ thymocytes, indicating that PD-L1 is involved in normal thymic selection. These data demonstrate that PD-1:PD-L1 interactions are critical to positive selection and play a role in shaping the T cell repertoire.
Animals↗
The Ras/MAPK cascade and the control of positive selection.
Immature double positive (DP) thymocytes bearing a T cell receptor (TCR) that interacts with self-major histocompatibility complex (MHC) molecules receive signals that induce either their differentiation (positive selection) or apoptosis (negative selection). Furthermore, those cells that are positively selected develop into two different lineages, CD4 or CD8, depending on whether their TCRs bind to MHC class II or I, respectively. Positive selection therefore involves rescue from the default fate (death), lineage commitment, and progression to the single positive (SP) stage. These are probably temporally distinct events that may require both unique and overlapping signals. Work in the past several years has started to unravel the signaling networks that control these processes. One of the first pathways identified as important for positive selection was Ras and its downstream effector, the Erk mitogen-activated protein kinase (MAPK) cascade. In this review we examine the factors that connect the TCR to the Ras/Erk cascade in DP thymocytes, as well as what we know about the downstream effectors of the Ras/Erk cascade important for positive selection. We also consider the possible role of this cascade in CD4/CD8 lineage development, and the possible interactions of the Ras/Erk cascade with Notch during these cell fate determination processes.
Animals↗
Reactivation of the ATCase domain of the URA2 gene complex: a positive selection method for Ty insertions and chromosomal rearrangements in Saccharomyces cerevisiae.
Genetic rearrangements such as deletions or duplications of DNA sequences are rarely detected in the yeast Saccharomyces cerevisiae. We have developed a screening system using the URA2 gene coding for the bi-functional CPSase-ATCase (carbamyl phosphate synthetase - aspartate transcarbamylase) to select positively for these kinds of events. Nonsense mutations in the CPSase region cause a complete loss of the ATCase activity because of their strong polar effect. Thirty-seven ATCase+ revertants were isolated from a strain containing three nonsense mutations in the proximal CPSase region. Genetic and structural analysis of the URA2 locus in these strains allowed us to characterize two major classes of revertants. In the first, an entire copy of a Ty transposon was found to be inserted in the CPSase coding domain. This event, which represents a new form of Ty-mediated gene activation was further analysed by mapping the Ty integration site in 26 strains. In a second class of revertants, we observed chromosomal rearrangements and, in particular, duplication of the ATCase region and its integration in a new chromosomal environment in which this sequence becomes active.
Aspartate Carbamoyltransferase↗
Expression of the Eco RI restriction-modification system and the construction of positive-selection cloning vectors.
The genes encoding the Eco RI restriction-modification (R/M) system have been separately cloned onto compatible plasmids. We have shown that the Eco RI restriction gene is expressed in the total absence of methylase enzyme and confirmed that a temperature-sensitive mutant is defective in Eco RI modification activity at higher temperatures. Insertion of transcriptional terminators into the restriction gene had no detectable effect on Eco RI modification activity. This strongly suggests that a separate promoter exists for the methylase gene. Analysis of the published sequence shows that the methylase gene promoter may overlap with the COOH-terminal region of the endonuclease structural gene. The temperature-sensitive Eco RI system has been exploited in the construction of two plasmid cloning vectors, pLV57 and pLV59, which can be used to select positively for transformants bearing recombinant plasmids; cloning of a DNA fragment into pLV57 or pLV59 at the unique HindIII, Bg/II, or PstI sites inactivates the Eco RI restriction gene and permits the hybrid plasmid to survive at 37 degrees C. The temperature-sensitive modification activity of these vectors should also facilitate the introduction of Eco RI linkers into DNA cloned in this way.
Base Sequence↗
Differences in the avidity of TCR interactions with a superantigenic ligand affect negative selection but do not allow positive selection.
The products of the sag genes of the exogenous mouse mammary tumor virus (MMTV) genome and of endogenous Mtv integrants have been demonstrated to affect the T cell repertoire in mice by causing the deletion of T cells expressing receptors encoded by particular V beta gene segments. Since these deletions affect large populations of T cells with receptors of heterogeneous specificity, they serve as an important model for the study of T cell development in normal mice. Using several C3H/HeN-based strains that express different MMTV(C3H) transgenes, we demonstrate here that the stage of development at which T cell deletion occurs is determined by the level of ligand expression. Although at low levels of ligand expression in the thymus some signs of activation were observed in immature thymocytes, we were unable to detect a level of Sag expression that led to net positive selection. Moreover, we detected a level of Sag-transgene expression that did not cause negative selection in the thymus; no signs of positive selection were observed either. Inclusion of the env gene in the construct, earlier shown to markedly potentiate stimulation by Sag in mixed lymphocyte reactions, also markedly increased the ability of Sag to drive negative selection. These data are interpreted as showing that marked quantitative differences in expression of superantigens does not reveal a level at which only positive selection occurs. This, in turn, suggests that positive selection will occur on ligands distinct from those that drive clonal deletion.
Aging↗
Positive selection of Tac- (CD25) positive cells following T-cell activation. Use of immunomagnetic separation and implications for T-cell cloning.
We have investigated if positive selection for cells expressing activation antigens, which appear on the cell surface during T-lymphocyte activation, could be used for cloning purposes. For this purpose, we used paramagnetic, monodisperse Dynabeads coated with anti-Tac monoclonal antibody, which recognizes CD25 (interleukin-2 receptor light chain). After the first 6-12 h of a primary response, depletion of Tac+ cells could largely abrogate the specific response. This indicated that the specifically responding cells were found among the Tac+ population. T-cell cloning was thus performed on Tac+ blasts positively selected after 18 h of a primary response, at day 6 of a primary response or during secondary stimulation, and gave a high percentage of specific clones. This method is thus a good alternative to established techniques.
Cell Separation↗
Facilitation of beta selection and modification of positive selection in the thymus of PD-1-deficient mice.
PD-1 is an immunoglobulin superfamily member bearing an immunoreceptor tyrosine-based inhibitory motif, and disruption of the PD-1 gene results in the development of lupus-like autoimmune diseases. In this study, we examined effects of the PD-1 deficiency on the thymocyte differentiation at the clonal level using T cell receptor (TCR)-beta (Vbeta8) and TCR-alpha/beta (H-Y and 2C) transgenic mice. In these TCR transgenic lines, PD-1 expression in the thymus was variably augmented, but as in the normal mice, confined largely to the CD4(-)CD8(-) thymocytes. The transgenic mice crossed with PD-1(-/)- mice in the neutral genetic backgrounds exhibited selective increase in the CD4(+)CD8(+) (DP) population with little effect on other thymocytes subsets. Similarly, the absence of PD-1 facilitated expansion of DP thymocytes in recombination activating gene (RAG)-2(-/)- mice by anti-CD3epsilon antibody injection. On the other hand, H-Y or 2C transgenic PD-1(-/)- mice with the positively selecting background showed significantly reduced efficiency for the generation of CD8(+) single positive cells bearing the transgenic TCR-alpha/beta in spite of the increased DP population. These results collectively indicate that PD-1 negatively regulates the beta selection and modulates the positive selection, and suggest that PD-1 deficiency may lead to the significant alteration of mature T cell repertoire.
Animals↗
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.
Animals↗
Improvement of tumor cell depletion by combining immunomagnetic positive selection of CD34-positive hematopoietic stem cells and negative selection (purging) of tumor cells.
One possible reason for relapse after high-dose chemotherapy is retransplantation of tumor cells contaminating autologous hematopoietic stem cell transplants. Residual tumor cells can be diminished by various purging methods. We studied tumor cell depletion by sequentially combining immunomagnetic positive selection of CD34+ hematopoietic stem cells using Isolex50 or Isolex300SA and negative tumor cell depletion using MACS, MaxSep or Isolex50 systems. Using these separation systems in different selection sequences, i.e. positive followed by negative selection (+/- selection) or vice versa, four groups of double selections (Isolex50/MACS, Isolex50/MaxSep, MaxSep/Isolex50, Isolex300SA/Isolex50) were studied. Testing these double-purging procedures mean additional tumor cell depletion (deltaTCD) achieved by the second selection step ranged from 1.1+/-0.58 log (n = 5, +/- Isolex50/MACS) to 2.0+/-1.1 log (n = 7, -/+ MaxSep/Isolex50). Loss of CD34+ cells during double selection sometimes was extensive and mean yield of CD34+ cells ranged from 12.8+/-11.5% (n = 6, +/- Isolex50/MaxSep) to 43.2% (n = 2, +/- Isolex300SA/Isolex50). Calculated values for mean yield-corrected deltaTCD ranged from 0.64+/-0.3 log (n = 5, +/- Isolex50/MACS) to 1.4+/-1.3 log (n = 7, -/+ MaxSep/Isolex50). During positive selection of -/+ selection (MaxSep/Isolex50) relative tumor cell enrichment was detectable leading to an increment of mean tumor cell contamination rate. Best results for total TCD were achieved by the combination of Isolex50/MaxSep (n = 6; TCD: 4.2 log; yield CD34+: 12.8%) and Isolex300/Isolex50 (n = 2; TCD: 3.8 log; yield CD34+: 43.2%). Furthermore, we have established and tested a new simultaneous +/- selection method by using CD34-specific releasing agent PR34+ in the Isolex300i. With this method we have obtained a mean total yield-corrected TCD of 4.7 log (n = 4; range: 4.1-6.0 log) with high CD34+ cell yield (mean: 69.8%) and CD34+ cell purity (mean: 92.8%). Since this new simultaneous +/- purging procedure is safe, applicable within a closed system (GMP-like) and most effective, we recommend it for further testing in a clinical setting.
Antigens, CD34↗