scholarly journals The Developmentally Regulated Expression of Twisted Gastrulation Reveals a Role for Bone Morphogenetic Proteins in the Control of T Cell Development

2002 ◽  
Vol 196 (2) ◽  
pp. 163-171 ◽  
Author(s):  
Daniel Graf ◽  
Suran Nethisinghe ◽  
Donald B. Palmer ◽  
Amanda G. Fisher ◽  
Matthias Merkenschlager
Keyword(s):  
T Cell ◽  
Bone Morphogenetic Proteins ◽  
Cell Receptor ◽  
Mammalian Development ◽  
Double Positive ◽  
Developmentally Regulated ◽  
Twisted Gastrulation ◽  
Thymocyte Proliferation ◽  
Regulated Expression

The evolutionarily conserved, secreted protein Twisted gastrulation (Tsg) modulates morphogenetic effects of decapentaplegic (dpp) and its orthologs, the bone morphogenetic proteins 2 and 4 (BMP2/4), in early Drosophila and vertebrate embryos. We have uncovered a role for Tsg at a much later stage of mammalian development, during T cell differentiation in the thymus. BMP4 is expressed by thymic stroma and inhibits the proliferation of CD4−CD8− double-negative (DN) thymocytes and their differentiation to the CD4+CD8+ double-positive (DP) stage in vitro. Tsg is expressed by thymocytes and up-regulated after T cell receptor signaling at two developmental checkpoints, the transition from the DN to the DP and from the DP to the CD4+ or CD8+ single-positive stage. Tsg can synergize with the BMP inhibitor chordin to block the BMP4-mediated inhibition of thymocyte proliferation and differentiation. These data suggest that the developmentally regulated expression of Tsg may allow thymocytes to temporarily withdraw from inhibitory BMP signals.

scholarly journals Contributions of the T Cell Receptor–associated CD3γ–ITAM to Thymocyte Selection

2002 ◽  
Vol 196 (1) ◽  
pp. 1-13 ◽  
Author(s):  
Mariëlle C. Haks ◽  
Elsa Pépin ◽  
Jeroen H.N. van den Brakel ◽  
Sigrid A.A. Smeele ◽  
Stanley M. Belkowski ◽  
...  
Keyword(s):  
T Cell ◽  
Positive Selection ◽  
T Cell Receptor ◽  
Dissociation Constants ◽  
Cell Receptor ◽  
Double Positive ◽  
Mutant Receptors

The immunoreceptor tyrosine-based activation motifs (ITAMs) in the CD3 chains associated with the T cell receptor (TCR) are crucial for TCR signaling. To probe the role of the CD3γ–ITAM in T cell development, we created knock-in mice in which the CD3γ chain of the TCR complex is replaced by a mutant signaling-deficient CD3γ chain, lacking the CD3γ–ITAM. This mutation results in considerable impairment in positive selection in the polyclonal TCR repertoire. When CD3γ–ΔITAM mice are crossed to mice expressing transgenic F5 TCRs, their thymocytes are completely unable to perform positive selection in vivo in response to intrathymic ligands. Also, the in vitro positive selection response of double-positive (DP) thymocytes with F5–CD3γ–ΔITAM mutant receptors to their agonist ligand and many of its variants is severely impaired or abrogated. Yet, the binding and dissociation constants of agonist ligands for the F5 receptor are not affected by the CD3γ–ΔITAM mutation. Furthermore, DP thymocytes with mutant receptors can respond to agonist ligand with normal antigen sensitivity and to normal levels, as shown by their ability to induce CD69 up-regulation, TCR down-regulation, negative selection, and ZAP70 and c-Jun NH2-terminal kinase activation. In sharp contrast, induction of extracellular signal-regulated kinase (ERK) activation and linker for activation of T cells (LAT) phosphorylation are severely impaired in these cells. Together, these findings underscore that intrinsic properties of the TCR–CD3 complex regulate selection at the DP checkpoint. More importantly, this analysis provides the first direct genetic evidence for a role of the CD3γ–ITAM in TCR-driven thymocyte selection.

scholarly journals In Vitro Negative Selection of Viral Superantigen-Reactive Thymocytes by Thymic Dendritic Cells

Blood ◽  
1997 ◽  
Vol 90 (5) ◽  
pp. 1943-1951 ◽  
Author(s):  
Isabel Ferrero ◽  
Fabienne Anjuère ◽  
H. Robson MacDonald ◽  
Carlos Ardavı́n
Keyword(s):  
Dendritic Cells ◽  
Experimental System ◽  
Cell Receptor ◽  
Double Positive ◽  
Clonal Deletion ◽  
Vitro Assay ◽  
Thymocyte Proliferation ◽  
Tumor Virus

Abstract Intrathymic expression of endogenous mouse mammary tumor virus (MMTV)–encoded superantigens (SAg) induces the clonal deletion of T cells bearing SAg-reactive T-cell receptor (TCR) Vβ elements. However, the identity of the thymic antigen-presenting cells (APC) involved in the induction of SAg tolerance remains to be defined. We have analyzed the potential of dendritic cells (DC) to mediate the clonal deletion of Mtv-7-reactive TCR αβ P14 transgenic thymocytes in an in vitro assay. Our results show that both thymic and splenic DC induced the deletion of TCR transgenic double positive (DP) thymocytes. DC appear to be more efficient than splenic B cells as negatively selecting APC in this experimental system. Interestingly, thymic and splenic DC display a differential ability to induce CD4+SP thymocyte proliferation. These observations suggest that thymic DC may have an important role in the induction of SAg tolerance in vivo.

scholarly journals T cell receptor-mediated selection of functional rat CD8 T cells from defined immature thymocyte precursors in short-term suspension culture.

1991 ◽  
Vol 173 (3) ◽  
pp. 561-568 ◽  
Author(s):  
T Hünig ◽  
R Mitnacht
Keyword(s):  
T Cells ◽  
T Cell ◽  
T Cell Receptor ◽  
Cytotoxic T Lymphocyte ◽  
Ex Vivo ◽  
Interleukin 2 ◽  
Cell Receptor ◽  
Double Positive ◽  
Homogeneous Population

Recent results have indicated that positive and negative repertoire selection act on the major population of CD4,8 double-positive (DP) thymocytes that express 5-10-fold less T cell receptor (TCR) than mature T cells (i.e., they are TCRlow). Since DP cells obtained ex vivo are heterogeneous with regard to their stage within thymic selection, a homogeneous population of virgin DP cells suitable for selection studies was generated in vitro from their immediate precursors, the CD8 single-positive (SP) immature blast cells. To mimic TCR-mediated selection signals, these virgin DP cells were then cultured for another 2 d in the presence of immobilized anti-TCR monoclonal antibodies with or without interleukin 2 (IL-2). Daily monitoring of recovery and phenotype showed that without TCR stimulation, the cells remained DP and became small, TCRlow cells that were lost with a half-life of 1 d, regardless of the presence of IL-2. TCR stimulation resulted in rapid downregulation of CD4 and CD8, maintenance of a larger cell size, and induction of the CD53 antigen that marks mature and CD4,8 double-negative rat thymocytes. In the absence of IL-2, viability decreased as rapidly as without TCR stimulation. Addition of IL-2 rescued TCR-stimulated virgin DP cells and prevented CD8 downregulation, so that 50-80% of input DP cells were recovered after 2 d as CD4-8+53+ cells. After release from modulation, these in vitro generated CD8 SP cells quantitatively upregulated the TCR to the TCRhigh phenotype and were readily induced to proliferate and exhibit cytotoxic T lymphocyte (CTL) activity in a polyclonal readout. Evidence is presented implicating an IL-2 receptor (IL-2R) not containing the p55 chain (i.e., most likely the p70 intermediate affinity IL-2R) in the TCR plus IL-2-driven in vitro differentiation of virgin DP cells towards the mature CD8 SP phenotype.

scholarly journals T cell receptor ligation induces interleukin (IL) 2R beta chain expression in rat CD4,8 double positive thymocytes, initiating an IL-2-dependent differentiation pathway of CD8 alpha+/beta- T cells.

1993 ◽  
Vol 177 (2) ◽  
pp. 541-546 ◽  
Author(s):  
J H Park ◽  
R Mitnacht ◽  
N Torres-Nagel ◽  
T Hünig
Keyword(s):  
T Cells ◽  
T Cell ◽  
T Cell Receptor ◽  
Cd8 T Cells ◽  
Cell Receptor ◽  
Beta Chain ◽  
Double Positive ◽  
Alpha Chain ◽  
Alpha Beta

The role of interleukin (IL)2 in intrathymic T cell development is highly controversial, and nothing is known about IL-2R expression on thymocytes of the T cell receptor (TCR) alpha/beta lineage undergoing TCR-driven differentiation events. We analyze here IL-2R alpha and beta mRNA expression in an in vitro system where newly generated rat CD4,8 double positive (DP) thymocytes respond to TCR ligation plus IL-2 (but not to either stimulus alone) with rapid differentiation to functional CD8 single positive T cells (Hünig, T., and R. Mitnacht. 1991. J. Exp. Med. 173:561). TCR ligation induced expression of IL-2R beta (but not alpha) chain mRNA in DP thymocytes. Addition of IL-2 then lead to functional maturation and expression of the IL-2R alpha chain. To investigate if the CD8 T cells generated via this IL-2R beta-driven pathway in vitro correspond to the bulk of CD8 T cells seeding peripheral lymphoid organs in vivo, we compared their phenotype to that of lymph node CD8 T cells. Surprisingly, analysis of CD8 cell surface expression using a novel anti-CD8 monoclonal antibody specific for the alpha/beta heterodimeric isoform, and of CD8 alpha and beta chain mRNA revealed that T cells generated by TCR ligation plus IL-2 resemble thymus-independent rather than thymus-derived CD8 cells in that they express CD8 alpha without beta chains. These findings demonstrate that TCR crosslinking induces functional IL-2R on immature DP rat thymocytes. In addition, they show that at least in vitro, CD8 alpha/alpha T cells are generated from TCR-stimulated DP thymocytes (which express the CD8 alpha/beta in the heterodimeric isoform) along an IL-2-driven pathway of T cell differentiation.

scholarly journals CD6 modulates thymocyte selection and peripheral T cell homeostasis

2016 ◽  
Vol 213 (8) ◽  
pp. 1387-1397 ◽  
Author(s):  
Marc Orta-Mascaró ◽  
Marta Consuegra-Fernández ◽  
Esther Carreras ◽  
Romain Roncagalli ◽  
Amado Carreras-Sureda ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Immunological Synapse ◽  
Cell Receptor ◽  
Autoimmune Response ◽  
Effector Memory ◽  
Thymocyte Development ◽  
Double Positive

The CD6 glycoprotein is a lymphocyte surface receptor putatively involved in T cell development and activation. CD6 facilitates adhesion between T cells and antigen-presenting cells through its interaction with CD166/ALCAM (activated leukocyte cell adhesion molecule), and physically associates with the T cell receptor (TCR) at the center of the immunological synapse. However, its precise role during thymocyte development and peripheral T cell immune responses remains to be defined. Here, we analyze the in vivo consequences of CD6 deficiency. CD6−/− thymi showed a reduction in both CD4+ and CD8+ single-positive subsets, and double-positive thymocytes exhibited increased Ca2+ mobilization to TCR cross-linking in vitro. Bone marrow chimera experiments revealed a T cell–autonomous selective disadvantage of CD6−/− T cells during development. The analysis of TCR-transgenic mice (OT-I and Marilyn) confirmed that abnormal T cell selection events occur in the absence of CD6. CD6−/− mice displayed increased frequencies of antigen-experienced peripheral T cells generated under certain levels of TCR signal strength or co-stimulation, such as effector/memory (CD4+TEM and CD8+TCM) and regulatory (T reg) T cells. The suppressive activity of CD6−/− T reg cells was diminished, and CD6−/− mice presented an exacerbated autoimmune response to collagen. Collectively, these data indicate that CD6 modulates the threshold for thymocyte selection and the generation and/or function of several peripheral T cell subpopulations, including T reg cells.

scholarly journals The concurrent maturation of mouse and human thymocytes in human fetal thymus implanted in NIH-beige-nude-xid mice is associated with the reconstitution of the murine immune system.

1993 ◽  
Vol 177 (3) ◽  
pp. 821-832 ◽  
Author(s):  
T R Kollmann ◽  
M M Goldstein ◽  
H Goldstein
Keyword(s):  
T Cells ◽  
Immune System ◽  
T Cell ◽  
Keyhole Limpet Hemocyanin ◽  
Cell Receptor ◽  
Scid Mice ◽  
Double Positive ◽  
Fetal Thymus

To determine whether the human thymus provides an environment for the maturation of murine T cells, human fetal thymus and liver (hu-thy/liv) were implanted into congenitally athymic NIH-beige-nude-xid (BNX) mice or C.B-17 scid/scid (SCID) mice. 3 mo after implantation, in contrast to the hu-thy/liv implant in SCID mice, which was populated only with human CD4/CD8 single- and double-positive thymocytes, the hu-thy/liv implant in BNX mice contained a chimeric population of human and mouse CD4/CD8 single- and double-positive thymocytes. Immunohistochemical staining of the hu-thy/liv implant in BNX mice indicated that the population of double-positive mouse thymocytes was localized to discrete areas of the human fetal thymus. Quantitative improvements in mouse T cell and immunoglobulin (Ig) G parameters were observed after grafting of the human fetal thymus and liver tissue into BNX mice. In addition, in contrast to the nonimplanted BNX mice, the implanted BNX mice were capable of mounting a keyhole limpet hemocyanin-specific IgG response and their peripheral T cells were responsive to stimulation with mitogens and antibodies directed to the T cell receptor. Furthermore, after in vivo priming, T cells present in lymph nodes of the implanted BNX mice were capable of mounting an antigen-induced in vitro T cell-dependent proliferative response. Thus, concurrent with the continued maturation of human T cells, murine T cells differentiated within the human fetal thymus implanted in the BNX mice and mediated the phenotypic and functional reconstitution of the murine immune system. Mice with a reconstituted immune system that contain a human thymic implant that is infectible with human immunodeficiency virus (HIV) should prove useful in the investigation of T cell maturation in the thymus and in the evaluation of potential HIV vaccines.

Essential role of Rap signal in pre-TCR–mediated β-selection checkpoint in αβ T-cell development

Blood ◽  
2008 ◽  
Vol 112 (12) ◽  
pp. 4565-4573 ◽  
Author(s):  
Kohei Kometani ◽  
Masaki Moriyama ◽  
Yasuhiro Nakashima ◽  
Yoshinori Katayama ◽  
Shu-Fang Wang ◽  
...  
Keyword(s):  
T Cell ◽  
Essential Role ◽  
T Cell Development ◽  
Cell Receptor ◽  
Cell Development ◽  
Dominant Negative ◽  
Nucleotide Exchange ◽  
Double Positive ◽  
Conditional Expression

Abstract We demonstrate that lck promoter–driven conditional expression of transgenic SPA-1, a Rap GTPase-activation protein, causes a profound defect of αβ T-cell development at the CD4/CD8 double-negative (DN) stage due to enhanced cell death without affecting γδ T-cell development. The effect was specific to the DN stage, because CD4 promoter–driven SPA-1 expression hardly affected T-cell development. Rap1A17, a dominant-negative Rap mutant, interfered with the generation of double-positive (DP) cells from Rag2−/− fetal thymocytes in vitro in the presence of anti-CD3ϵ antibody and Notch ligand. Rap GTPases were activated in a DN cell line by the expression of self-oligomerizing CD3 (CD8:CD3ϵ chimera), which substituted autonomous pre–T-cell receptor (TCR) signal, inducing CD69 expression and CD25 down-regulation. Reciprocally, expression of C3G, a Rap guanine nucleotide exchange factor, in both normal and Rag2−/− DN cells markedly enhanced Notch-dependent generation and expansion of DP cells without additional anti-CD3ϵ antibody, thus bypassing pre-TCR. Defective αβ T-cell development in the conditional SPA-1–transgenic mice was restored completely by introducing a p53−/− mutation. These results suggest that endogenous Rap GTPases downstream of pre-TCR play an essential role in rescuing pre-T cells from the p53-mediated checkpoint response, thus allowing Notch-mediated expansion and differentiation.

scholarly journals p56lck Signals for Regulating Thymocyte Development Can Be Distinguished by Their Dependency on Rho Function

1998 ◽  
Vol 188 (5) ◽  
pp. 931-939 ◽  
Author(s):  
Stefan W. Henning ◽  
Doreen A. Cantrell
Keyword(s):  
T Cells ◽  
T Cell ◽  
Rho Gtpase ◽  
Antigen Expression ◽  
Cell Receptor ◽  
Thymocyte Development ◽  
Double Positive ◽  
Thymocyte Proliferation ◽  
Function Expression ◽  
And Control

The tyrosine kinase p56lck regulates the differentiation and proliferative expansion of pre-T cells. However, nothing is known about other signaling molecules that operate with p56lck to mediate the pleiotropic changes that occur at this stage of thymocyte development. We used a genetic strategy to examine the requirement for the GTPase Rho in p56lck-mediated signals in the thymus. By generating mice double transgenic for a constitutively activated form of p56lck (p56lckF505) and the Rho inhibitor C3 transferase we were able to compare thymocyte development in mice expressing active p56lck on a wild-type or Rho− background. Thymocytes expressing active p56lck show enhanced proliferation of pre-T cells resulting in increased numbers of late pre-T cells, however, this dramatic effect on pre-T cell proliferation is lost when the p56lck transgene is expressed in thymocytes lacking endogenous Rho GTPase function. Expression of active p56lck also generates double positive (DP) thymocytes with low levels of CD2 antigen expression. Again, p56lck cannot prevent expression of CD2 when expressed on a Rho− background. CD4+CD8+ DP cells expressing active p56lck have been shown to lack functional α/β–T cell receptor (TCR) complexes due to p56lck-mediated inhibition of TCR gene Vβ-Dβ rearrangement. This inhibition of TCR expression by active p56lck is unimpaired in the absence of Rho function. The signaling pathways that are mediated by p56lck and control thymocyte proliferation, α/β-TCR and CD2 antigen expression can thus be distinguished by their dependency on Rho function.

scholarly journals Mechanisms of tolerance induction in major histocompatibility complex class II-restricted T cells specific for a blood-borne self-antigen.

1994 ◽  
Vol 180 (6) ◽  
pp. 2089-2099 ◽  
Author(s):  
T Zal ◽  
A Volkmann ◽  
B Stockinger
Keyword(s):  
T Cell ◽  
Transgenic Mice ◽  
T Cell Receptor ◽  
Tolerance Induction ◽  
Cell Receptor ◽  
Double Positive ◽  
Single Positive ◽  
Self Antigen

Transgenic mice expressing a major histocompatibility complex class II-restricted T cell receptor with specificity for a natural self-antigen, the fifth component of complement, were generated to analyze the mechanism of tolerance induction to a blood-borne self-protein. In the absence of C5 protein thymocytes from T cell receptor transgenic mice develop into mature CD4 single positive cells which emigrate into the periphery and mount C5-specific T cell responses upon immunization with C5. In the presence of circulating C5 protein, CD4 single positive thymocytes do not develop. Negative selection occurs late in thymic ontogeny leaving the bulk of CD4+8+ thymocytes unaffected. This phenotype may be due to a delay in contact with self-antigen presentation which, under physiological conditions, is inefficient in the cortex of C5+ mice, and therefore does not affect most immature double positive thymocytes. In contrast, in vitro exposure to C5(-)-presenting dendritic cells or in vivo injection of C5 peptide results in deletion of double positive thymocytes. C5+ transgenic mice are tolerant in vivo, but contain T cells in spleen and lymph nodes that secrete interleukin 2 and interferon gamma in response to C5 activation in vitro. When crossed onto a Rag1-/- background to prevent endogenous T cell receptor rearrangements, these peripheral potentially autoreactive cells do not appear. This indicates that endogenous T cell receptor rearrangements possibly leading to the expression of two receptors might be a prerequisite for their survival and export into the periphery.

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