scholarly journals Lineage relationships and developmental kinetics of immature thymocytes: CD3, CD4, and CD8 acquisition in vivo and in vitro.

1990 ◽  
Vol 172 (6) ◽  
pp. 1583-1588 ◽  
Author(s):  
H T Petrie ◽  
P Hugo ◽  
R Scollay ◽  
K Shortman
Keyword(s):  
Direct Injection ◽  
Interleukin 2 ◽  
Cell Receptor ◽  
Developmental Sequence ◽  
Heat Stable Antigen ◽  
Single Positive ◽  
Kinetics Of ◽  
Immature Thymocytes

T lymphocytes develop in the thymus from immunologically naive bone marrow precursors. Based on T cell receptor rearrangement and transcription, and thymic reconstitution potential, we have deduced a developmental sequence among immature thymocytes, before the acquisition of the lineage markers CD3, CD4, and CD8. In the current study, we have followed the ontogenic progression of the latter stages in this sequence, using two different systems: (a) in vivo, by direct injection into the thymus of nonirradiated, congenic recipients; and (b) in vitro, using culture medium without mitogens or cytokines. In vivo, the less mature Pgp-1- interleukin 2 receptor alpha-positive (IL-2R alpha+) CD3-4-8- subset (also heat-stable antigen high) requires 3 d before becoming predominantly IL-2R alpha- CD3lo4+ 8+ typical cortical-type cells, and at least 5 d before the appearance of any mature single-positive cells (CD3hi4+ 8- or CD3hi4-8+). However, these Pgp-1- IL-2R alpha+ precursors do not differentiate further in unstimulated culture. The more mature Pgp-1- IL-2R alpha- CD3-4-8- subset becomes primarily CD3lo4+ 8+ within 1 d after transplantation, and some mature single-positive progeny are evident by day 3. By 5 d, most of these Pgp-1-IL-2R alpha- precursor cells have become CD3hi, and have lost or are downregulating either CD4 or CD8. In culture, these Pgp-1- IL-2R alpha- cells also acquire high levels of CD4 and CD8 within 1 d, and low levels of CD3 by 2 d. However, they do not progress further to mature single positives in vitro, and most of them die by day 3. These experiments directly confirm our previously proposed developmental sequence, and demonstrate the kinetics of T lymphocyte production in a low-stress, steady-state environment.

Functional and Molecular Analysis of Maturation of Thymocytes in Bcl10 or Malt1 Deficient Mice.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 3323-3323
Author(s):  
Philipp J. Jost ◽  
Uta Ferch ◽  
Stephanie Weiss ◽  
Stephanie Leeder ◽  
Olaf Gross ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Transgenic Animals ◽  
Adaptor Proteins ◽  
Cell Receptor ◽  
Single Positive ◽  
T Cell Selection ◽  
Deficient Mice

Abstract Development of immature T cells in the thymus requires signals through the clonotypic T cell receptor (TCR). Thymocytes expressing a functionally inactive or autoreactive TCR are deleted via apoptosis (negative selection). Thymocytes expressing a functionally active but not autoreactive TCR are selected through inhibition of cell death (positive selection). Deregulation of this process is likely to result in autoimmunity or lymphomagenesis of T cells. The intracellular mechanisms by which the balance between TCR-dependent survival and apoptosis are regulated are largely unknown. A central regulator of survival and apoptosis in the immune system is the transcription factor NF-κB. Activation of NF-κB in mature T-cells requires the adaptor proteins Bcl10 and Malt1. Using gene-targeted mice deficient for Bcl10 or Malt1, we show that Bcl10 and Malt1 are also required for TCR-induced NF-κB activation in immature T cells. Furthermore, to elucidate the process of T cell selection within the thymus, we have crossed Bcl10 or Malt1 deficient mice into mice with genetic backgrounds expressing defined TCR transgenes. Using specific peptide agonists of these TCR transgenes, we show that neither in vivo nor in vitro development into single positive (SP) CD4 or CD8 positive T cells is altered in Bcl10 or Malt1 deficient mice. Absolute numbers and ratio of SP T cells found within the thymus or in peripheral lymphnodes of transgenic animals are normal. These findings indicate that Bcl10 and Malt1 activate NF-κB in thymocytes but are dispensable for maturation of immature T cells in this model system.

scholarly journals Localization and phenotype of cycling and post-cycling murine thymocytes studied by simultaneous detection of bromodeoxyuridine and surface antigens.

1988 ◽  
Vol 36 (5) ◽  
pp. 473-478 ◽  
Author(s):  
C Penit
Keyword(s):  
T Cell ◽  
Interleukin 2 ◽  
Simultaneous Detection ◽  
Growth Period ◽  
Surface Antigens ◽  
Cell Receptor ◽  
Initial Growth ◽  
Outer Cortex

Bromodeoxyuridine (BrdUrd) was incorporated in vivo or in vitro into the DNA of proliferating murine thymocytes. Surface antigens Thy1, Lyt2 (CD8), L3T4 (CD4), interleukin-2 receptor (IL2-R), and the V beta 8 chain of the T-cell receptor were detected using specific monoclonal antibodies with the biotin-avidin system, and cells were then treated for DNA denaturation. Simultaneous detection of BrdUrd and surface markers was performed on cell smears and frozen sections by double-color immunofluorescence. The phenotype of cycling cells, determined in fetal thymus and in the thymus of mice from birth to one year of age, showed relative stability after the initial growth period, despite severe involution of the gland. Phenotypic evolution of cycling cells and their progeny was also studied in colchicine-treated animals and was shown to reproduce sequential events of T-cell differentiation. On sections, the highest frequency of cycling cells was observed in the outer cortex in normal thymus, but the first cells to start proliferation during regeneration were mostly located in the deep cortex and corticomedullary junction. These results show the high potential of this method, as compared to autoradiography of radiolabeled cells.

Thymic emigrants isolated by a new method possess unique phenotypic and functional properties

Blood ◽  
2001 ◽  
Vol 97 (5) ◽  
pp. 1360-1369 ◽  
Author(s):  
Chong-kil Lee ◽  
Kyungjae Kim ◽  
Lisbeth A. Welniak ◽  
William J. Murphy ◽  
Kathrin Muegge ◽  
...  
Keyword(s):  
T Cells ◽  
Severe Combined Immunodeficiency ◽  
Cell Receptor ◽  
Intercellular Adhesion Molecule ◽  
Graft Versus Host Reaction ◽  
Intercellular Adhesion Molecule 1 ◽  
Heat Stable Antigen ◽  
Heterogeneous Expression

T cells that emigrate from the thymus have primarily been studied in vivo using fluorescent dye injection of the thymus. This study examined the properties of thymocytes that emigrate from cultured thymic lobes in organ culture. Under these conditions, thymic emigrants displayed the expected phenotype, that of mature thymocytes expressing high levels of T-cell receptor (TCR-αβ) and either CD4 or CD8, and were observed to emigrate within 24 hours of positive selection. Emigration was inhibited by cytochalasin D, pertussis toxin, orClostridium difficile toxin B, implicating an active motility process. Most of the surface markers on αβ-thymic emigrants (Thy1, CD44, CD69, CD25, leukocyte functional antigen-1, intercellular adhesion molecule-1, α4-integrin, α5-integrin, CD45, and CD28) were expressed at a surface density similar to that on mature intrathymic cells and peripheral splenic T cells. Heterogeneous expression of L-selectin and heat-stable antigen (HSA) suggested that subsets emerge from the thymus with a commitment to different migration patterns. The only marker on emigrants not found on either intrathymic cells or mature spleen T cells was CTLA-4, which could dampen the response of emigrants to peripheral antigens. Antigen responsivenes measured in vitro against allogeneic dendritic cells showed a proliferative response comparable to that of splenic T cells. In vivo, however, thymic emigrants failed to induce an acute graft-versus-host reaction in allogeneic severe combined immunodeficiency recipients. This suggests that a mechanism operating in vivo, perhaps tolerance or migration pattern, attenuates the response of emigrants against antigens that did not induce their deletion in the thymus.

scholarly journals The fusion kinase ITK-SYK mimics a T cell receptor signal and drives oncogenesis in conditional mouse models of peripheral T cell lymphoma

2010 ◽  
Vol 207 (5) ◽  
pp. 1031-1044 ◽  
Author(s):  
Konstanze Pechloff ◽  
Julian Holch ◽  
Uta Ferch ◽  
Marc Schweneker ◽  
Kristina Brunner ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Tyrosine Kinase ◽  
T Cell Receptor ◽  
Interleukin 2 ◽  
Cell Receptor ◽  
Recurrent Event ◽  
Conditional Expression

Peripheral T cell lymphomas (PTCLs) are highly aggressive malignancies with poor prognosis. Their molecular pathogenesis is not well understood and small animal models for the disease are lacking. Recently, the chromosomal translocation t(5;9)(q33;q22) generating the interleukin-2 (IL-2)–inducible T cell kinase (ITK)–spleen tyrosine kinase (SYK) fusion tyrosine kinase was identified as a recurrent event in PTCL. We show that ITK-SYK associates constitutively with lipid rafts in T cells and triggers antigen-independent phosphorylation of T cell receptor (TCR)–proximal proteins. These events lead to activation of downstream pathways and acute cellular outcomes that correspond to regular TCR ligation, including up-regulation of CD69 or production of IL-2 in vitro or deletion of thymocytes and activation of peripheral T cells in vivo. Ultimately, conditional expression of patient-derived ITK-SYK in mice induces highly malignant PTCLs with 100% penetrance that resemble the human disease. Our work demonstrates that constitutively enforced antigen receptor signaling can, in principle, act as a powerful oncogenic driver. Moreover, we establish a robust clinically relevant and genetically tractable model of human PTCL.

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.

Impaired responsiveness to T-cell receptor stimulation and defective negative selection of thymocytes in CCR7-deficient mice

Blood ◽  
2007 ◽  
Vol 110 (13) ◽  
pp. 4351-4359 ◽  
Author(s):  
Ana C. M. Davalos-Misslitz ◽  
Tim Worbs ◽  
Stefanie Willenzon ◽  
Günter Bernhardt ◽  
Reinhold Förster
Keyword(s):  
Negative Selection ◽  
Cell Receptor ◽  
Calcium Flux ◽  
Double Positive ◽  
Activation Kinetics ◽  
Single Positive ◽  
Flux Response ◽  
Selection Of

The chemokine receptor CCR7 has been implicated in maintenance of thymus morphology and establishment of tolerance to self-antigens. In this study, we provide direct evidence that negative selection of maturing thymocytes is defective in CCR7-deficent mice. Impaired negative selection was observed after TCR/CD3 complex stimulation in vivo as well as in vitro and was prominent in both double-positive and semimature single positive cells (CD4+CD8−CD24high). It is noteworthy that thymocytes of CCR7−/− mice display defective negative selection in response to endogenous superantigens, demonstrating that the defect also occurs under physiological conditions. Disturbed negative selection was correlated with delayed activation kinetics and decreased calcium flux response of CCR7−/− thymocytes after in vitro TCR/CD3 stimulation, suggesting that an impaired response of CCR7−/− thymocytes via TCR-mediated signaling is responsible for defective negative selection in these mice.

Ultrastructure of melanocyte-keratinocyte interactions and pigment transfer in vitro

1978 ◽  
Vol 36 (2) ◽  
pp. 650-651
Author(s):  
Raul I. Garcia ◽  
Evelyn A. Flynn ◽  
George Szabo
Keyword(s):  
Direct Injection ◽  
Skin Pigmentation ◽  
Freeze Fracture ◽  
Pigment Granule ◽  
Time Lapse ◽  
Ultrastructural Level ◽  
Lanthanum Tracer ◽  
A Cell

Skin pigmentation in mammals involves the interaction of epidermal melanocytes and keratinocytes in the structural and functional unit known as the Epidermal Melanin Unit. Melanocytes(M) synthesize melanin within specialized membrane-bound organelles, the melanosome or pigment granule. These are subsequently transferred by way of M dendrites to keratinocytes(K) by a mechanism still to be clearly defined. Three different, though not necessarily mutually exclusive, mechanisms of melanosome transfer have been proposed: cytophagocytosis by K of M dendrite tips containing melanosomes, direct injection of melanosomes into the K cytoplasm through a cell-to-cell pore or communicating channel formed by localized fusion of M and K cell membranes, release of melanosomes into the extracellular space(ECS) by exocytosis followed by K uptake using conventional phagocytosis. Variability in methods of transfer has been noted both in vivo and in vitro and there is evidence in support of each transfer mechanism. We Have previously studied M-K interactions in vitro using time-lapse cinemicrography and in vivo at the ultrastructural level using lanthanum tracer and freeze-fracture.

Effect of pH and inhibitors on beta-amyloid fibril assembly

1996 ◽  
Vol 54 ◽  
pp. 752-753
Author(s):  
Beverly E. Maleeff ◽  
Timothy K. Hart ◽  
Stephen J. Wood ◽  
Ronald Wetzel
Keyword(s):  
Amyloid Fibril ◽  
Peptide Fragment ◽  
Hydrophobic Peptides ◽  
Amyloid Fibril Formation ◽  
Effects Of Ph ◽  
Severity Of The Disease ◽  
Kinetics Of ◽  
The Brain

Alzheimer's disease is characterized post-mortem in part by abnormal extracellular neuritic plaques found in brain tissue. There appears to be a correlation between the severity of Alzheimer's dementia in vivo and the number of plaques found in particular areas of the brain. These plaques are known to be the deposition sites of fibrils of the protein β-amyloid. It is thought that if the assembly of these plaques could be inhibited, the severity of the disease would be decreased. The peptide fragment Aβ, a precursor of the p-amyloid protein, has a 40 amino acid sequence, and has been shown to be toxic to neuronal cells in culture after an aging process of several days. This toxicity corresponds to the kinetics of in vitro amyloid fibril formation. In this study, we report the biochemical and ultrastructural effects of pH and the inhibitory agent hexadecyl-N-methylpiperidinium (HMP) bromide, one of a class of ionic micellar detergents known to be capable of solubilizing hydrophobic peptides, on the in vitro assembly of the peptide fragment Aβ.

Kinetics of Various 99mTc-Sn-Pyrophosphate Compounds in the Rat

1977 ◽  
Vol 16 (04) ◽  
pp. 157-162 ◽  
Author(s):  
C. Schümichen ◽  
B. Mackenbrock ◽  
G. Hoffmann
Keyword(s):  
Normal Saline ◽  
Half Life ◽  
Compound A ◽  
Short Half Life ◽  
Low Concentrations ◽  
The Stability ◽  
Kinetics Of ◽  
In Vitro Stability

SummaryThe bone-seeking 99mTc-Sn-pyrophosphate compound (compound A) was diluted both in vitro and in vivo and proved to be unstable both in vitro and in vivo. However, stability was much better in vivo than in vitro and thus the in vitro stability of compound A after dilution in various mediums could be followed up by a consecutive evaluation of the in vivo distribution in the rat. After dilution in neutral normal saline compound A is metastable and after a short half-life it is transformed into the other 99mTc-Sn-pyrophosphate compound A is metastable and after a short half-life in bone but in the kidneys. After dilution in normal saline of low pH and in buffering solutions the stability of compound A is increased. In human plasma compound A is relatively stable but not in plasma water. When compound B is formed in a buffering solution, uptake in the kidneys and excretion in urine is lowered and blood concentration increased.It is assumed that the association of protons to compound A will increase its stability at low concentrations while that to compound B will lead to a strong protein bond in plasma. It is concluded that compound A will not be stable in vivo because of a lack of stability in the extravascular space, and that the protein bond in plasma will be a measure of its in vivo stability.

Acquired Haemophilia: Functional Study of Antibodies to Factor VIII

1981 ◽  
Vol 45 (03) ◽  
pp. 285-289 ◽  
Author(s):  
J P Allain ◽  
A Gaillandre ◽  
D Frommel
Keyword(s):  
Factor Viii ◽  
Functional Study ◽  
Factor Viii Inhibitor ◽  
Acquired Haemophilia ◽  
Viii Inhibitor ◽  
Kinetics Of ◽  
Antibody Function ◽  
Native Plasma

SummaryFactor VIII complex and its interaction with antibodies to factor VIII have been studied in 17 non-haemophilic patients with factor VIII inhibitor. Low VIII:C and high VIIIR.Ag levels were found in all patients. VIII:WF levels were 50% of those of VTIIRrAg, possibly related to an increase of poorly aggregated and electrophoretically fast moving VIIIR:Ag oligomers.Antibody function has been characterized by kinetics of VIII :C inactivation, saturability by normal plasma and the slope of the affinity curve. Two major patterns were observed:1) Antibodies from 6 patients behaved similarly to those from haemophiliacs by showing second order inhibition kinetics, easy saturability and steep affinity slope (> 1).2) Antibodies from other patients, usually with lower titres, inactivated VIII :C according to complex order kinetics, were not saturable, and had a less steep affinity slope (< 0.7). In native plasma, or after mixing with factor VIII concentrate, antibodies of the second group did not form immune complexes with the whole factor VIII molecular complex. However, dissociation procedures did release some antibodies from apparently low molecular weight complexes formed in vivo or in vitro. For appropriate management of non-haemophilic patients with factor VIII inhibitor, it is important to determine the functional properties of their antibodies to factor VIII.

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