scholarly journals Kinetics of Thymocyte Subset Development and Selection Revealed by Cyclosporin A Treatment

1995 ◽  
Vol 4 (2) ◽  
pp. 117-126 ◽  
Author(s):  
Russell D.J. Huby ◽  
Ray Hicks ◽  
Lindsey K. Goff
Keyword(s):  
Flow Cytometry ◽  
Cyclosporin A ◽  
Positive Selection ◽  
Maximal Effect ◽  
Color Flow ◽  
Progressive Development ◽  
Single Positive ◽  
Autoreactive Cells ◽  
Kinetics Of ◽  
Immature Thymocytes

Cyclosporin A (CsA) inhibits the development of mature thymocytes from their CD4+CD8+precursors, but may allow autoreactive cells to mature. Using 3-color flow cytometry, we have followed the progressive development of thymocytes, including potentially autoreactive cells, during CsA treatment. Numbers of CD4+CD8+CD3highthymocytes dropped immediately, suggesting that the generation of these mature thymocyte precursors, normally dependent upon positive selection, was inhibited by CsA. Numbers of CD4-CD8+thymocytes also declined rapidly, but CD4-CD8+thymocytes were unaffected lfor 2 days, suggesting that the mature single-positive subsets are not symmetrically derived from a common GsA-sensitive precursor. An exceptional subset of CD8 SP thymocytes, expressing CD45RA, did not respond to CsA for about 10 days, indicating that they are distantly derived from a CsA-sensitive precursor. Apoptosis of TCR-Vβ3+thymocytes caused byMtυ-6, quantified according to the down-regulation of CD4 and CD8 on immature thymocytes, was partially inhibited by CsA, to maximal effect within 24 hours. This did not, however, facilitate their development into mature thymocytes.

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.

scholarly journals Thymic Medulla Epithelial Cells Acquire Specific Markers by Post-Mitotic Maturation

1996 ◽  
Vol 5 (1) ◽  
pp. 25-36 ◽  
Author(s):  
Claude Penit ◽  
Bruno Lucas ◽  
Florence Vasseur ◽  
Theresa Rieker ◽  
Richard L. Boyd
Keyword(s):  
Epithelial Cells ◽  
Marrow Cell ◽  
Lymphoid Cells ◽  
Color Flow ◽  
Normal Bone Marrow Cell ◽  
Undifferentiated Cells ◽  
Single Positive ◽  
Thymic Medulla ◽  
Kinetics Of ◽  
Medullary Cells

The development of thymocyte subsets and of the thymic epithelium in SCID and RAG-2-/– mice was monitored after normal bone-marrow-cell transfer. The kinetics of thymic reconstitution and their relationships with cell proliferation were investigated by using bromodeoxyuridine to detect DNA-synthesizing cells among lymphoid cells by 3-color flow cytometry, and in epithelial compartments by staining frozen sections. Thymocytes started to express CD8 and CD4 10 days after transfer, simultaneously with extensive proliferation. The first mature CD4+single-positive cells were generated, from resting CD4+CD8+cells after day 15. During this day 10–15 period, many epithelial cells positive for cortexspecific or panepithelial markers were labeled with BrdUrd after pulse-injection. Organized medullary epithelium also developed after day,15, that is, synchronously with the appearance of mature thymocytes, but medullary cells were never found BrdUrd+. These results suggest that, in these models, the reconstitution of the thymic epithelial network proceeds through expansion of preexisting cortical or undifferentiated cells and by later maturation (acquisition of specific markers) of medullary cells. This last process is dependent of the presence of mature thymocytes.

scholarly journals Neonatal castration affects intrathymic kinetics of T-cell differentiation and the spleen T-cell level

2007 ◽  
Vol 192 (3) ◽  
pp. 669-682 ◽  
Author(s):  
K Radojević ◽  
N Arsenović-Ranin ◽  
D Kosec ◽  
V Pešić ◽  
I Pilipović ◽  
...  
Keyword(s):  
T Cell ◽  
Negative Selection ◽  
Double Positive ◽  
Adult Rats ◽  
Single Positive ◽  
Disease Induction ◽  
Firm Basis ◽  
Thymocyte Differentiation ◽  
Autoreactive Cells ◽  
Kinetics Of

To test putative interdependence in the ontogenesis of the hypothalamic–pituitary–gonadal and thymic–lymphatic axes, thymocyte differentiation and maturation was examined in neonatally castrated (Cx) adult rats. In the hypercellular thymi of Cx rats, the proportion of the least mature CD4−CD8−TCRαβ− triple negative (TN) thymocytes was reduced, while the proportions of all downstream double positive (DP) subsets (TCRαβ−, TCRαβlow and TCRαβhigh) were increased when compared with neonatally sham-castrated (Sx) adult rats. This suggested an accelerated thymocyte transition from the TN to DP TCRαβlow developmental stage accompanied by an increased positive/ reduced negative thymocyte selection. The increased thymocyte surface density of Thy-1, which is implicated in thymocyte hyposensitivity to negative selection, in Cx rats further supports the previous assumption. The finding that the proportions of both single positive (SP) TCRαβhigh thymocyte subsets were reduced, while their numbers were increased (CD4+CD8−) or unaltered (CD4−CD8+), coupled with results demonstrating an increased level of CD4−CD8+ cells without changes in that of CD4+8− cells in the spleen indicate: (i) accelerated differentiation and maturation of the positively selected DP TCRαβhigh thymocytes towards CD4−8+ TCRαβhigh cells followed by increased emigration of the mature cells and (ii) decelerated differentiation and maturation towards CD4+8−TCRαβhigh cells in Cx rats. Furthermore, the unaltered proportion of intrathymically developing CD4+CD25+Foxp3+ regulatory cells in Cx rats, in light of putative hyposensitivity of thymocytes to negative selection suggesting reduced elimination of autoreactive cells, may provide a firm basis for understanding the reasons behind increased susceptibility of Cx rats to autoimmune disease induction.

scholarly journals OMIP 073: Analysis of human thymocyte development with a 14‐color flow cytometry panel

2021 ◽  
Author(s):  
Sarah‐Jolan Bremer ◽  
Laura Glau ◽  
Christina Gehbauer ◽  
Annika Boxnick ◽  
Daniel Biermann ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Thymocyte Development ◽  
Color Flow ◽  
Human Thymocyte

scholarly journals KD determination from time-resolved experiments on live cells with LigandTracer and reconciliation with end-point flow cytometry measurements

2021 ◽  
Author(s):  
Diana Spiegelberg ◽  
Jonas Stenberg ◽  
Pascale Richalet ◽  
Marc Vanhove
Keyword(s):  
Flow Cytometry ◽  
Live Cells ◽  
Time Resolved ◽  
Surface Receptors ◽  
Full Characterization ◽  
End Point ◽  
Titration Curves ◽  
Kinetics Of

AbstractDesign of next-generation therapeutics comes with new challenges and emulates technology and methods to meet them. Characterizing the binding of either natural ligands or therapeutic proteins to cell-surface receptors, for which relevant recombinant versions may not exist, represents one of these challenges. Here we report the characterization of the interaction of five different antibody therapeutics (Trastuzumab, Rituximab, Panitumumab, Pertuzumab, and Cetuximab) with their cognate target receptors using LigandTracer. The method offers the advantage of being performed on live cells, alleviating the need for a recombinant source of the receptor. Furthermore, time-resolved measurements, in addition to allowing the determination of the affinity of the studied drug to its target, give access to the binding kinetics thereby providing a full characterization of the system. In this study, we also compared time-resolved LigandTracer data with end-point KD determination from flow cytometry experiments and hypothesize that discrepancies between these two approaches, when they exist, generally come from flow cytometry titration curves being acquired prior to full equilibration of the system. Our data, however, show that knowledge of the kinetics of the interaction allows to reconcile the data obtained by flow cytometry and LigandTracer and demonstrate the complementarity of these two methods.

scholarly journals Kinetics of in vitro natural killer activity against K562 cells as detected by flow cytometry

Cytometry ◽  
1998 ◽  
Vol 32 (4) ◽  
pp. 280-285 ◽  
Author(s):  
Loris Zamai ◽  
Adriana R. Mariani ◽  
Giorgio Zauli ◽  
Luigi Rodella ◽  
Rita Rezzani ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Natural Killer ◽  
Natural Killer Activity ◽  
K562 Cells ◽  
Killer Activity ◽  
Kinetics Of

Immunophenotyping of T Lymphocytes by Three-Color Flow Cytometry in Healthy Newborns, Children, and Adults

1997 ◽  
Vol 84 (1) ◽  
pp. 46-55 ◽  
Author(s):  
Thomas W. McCloskey ◽  
Terri Cavaliere ◽  
Saroj Bakshi ◽  
Rita Harper ◽  
James Fagin ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
T Lymphocytes ◽  
Color Flow
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