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.

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 Human Cd25+Cd4+ T Regulatory Cells Suppress Naive and Memory T Cell Proliferation and Can Be Expanded in Vitro without Loss of Function

2001 ◽  
Vol 193 (11) ◽  
pp. 1295-1302 ◽  
Author(s):  
Megan K. Levings ◽  
Romina Sangregorio ◽  
Maria-Grazia Roncarolo
Keyword(s):  
T Cell ◽  
Peripheral Blood ◽  
T Regulatory Cells ◽  
Transforming Growth Factor ◽  
Cytotoxic T Lymphocyte ◽  
Cell Receptor ◽  
Major Advance ◽  
Loss Of Function

Active suppression by T regulatory (Tr) cells plays an important role in the downregulation of T cell responses to foreign and self-antigens. Mouse CD4+ Tr cells that express CD25 possess remarkable suppressive activity in vitro and in autoimmune disease models in vivo. Thus far, the existence of a similar subset of CD25+CD4+ Tr cells in humans has not been reported. Here we show that human CD25+CD4+ Tr cells isolated from peripheral blood failed to proliferate and displayed reduced expression of CD40 ligand (CD40L), in response to T cell receptor–mediated polyclonal activation, but strongly upregulated cytotoxic T lymphocyte–associated antigen (CTLA)-4. Human CD25+CD4+ Tr cells also did not proliferate in response to allogeneic antigen-presenting cells, but they produced interleukin (IL)-10, transforming growth factor (TGF)-β, low levels of interferon (IFN)-γ, and no IL-4 or IL-2. Importantly, CD25+CD4+ Tr cells strongly inhibited the proliferative responses of both naive and memory CD4+ T cells to alloantigens, but neither IL-10, TGF-β, nor CTLA-4 seemed to be directly required for their suppressive effects. CD25+CD4+ Tr cells could be expanded in vitro in the presence of IL-2 and allogeneic feeder cells and maintained their suppressive capacities. These findings that CD25+CD4+ Tr cells with immunosuppressive effects can be isolated from peripheral blood and expanded in vitro without loss of function represent a major advance towards the therapeutic use of these cells in T cell–mediated diseases.

Structural requirements for enhancement of T-cell responsiveness by the lymphocyte-specific tyrosine protein kinase p56lck

1992 ◽  
Vol 12 (6) ◽  
pp. 2720-2729
Author(s):  
L Caron ◽  
N Abraham ◽  
T Pawson ◽  
A Veillette
Keyword(s):  
T Cell ◽  
Protein Phosphorylation ◽  
Phospholipase C ◽  
Interleukin 2 ◽  
Cell Receptor ◽  
Amino Terminal ◽  
Src Homology ◽  
Phospholipase C Gamma ◽  
Domain 3

To understand the mechanism(s) by which p56lck participates in T-cell receptor (TCR) signalling, we have examined the effects of mutations in known regulatory domains of p56lck on the ability of F505 p56lck to enhance the responsiveness of an antigen-specific murine T-cell hybridoma. A mutation of the amino-terminal site of myristylation (glycine 2), which prevents stable association of p56lck with the plasma membrane, completely abolished the ability of F505 p56lck to enhance TCR-induced tyrosine protein phosphorylation. Alteration of the major site of in vitro autophosphorylation, tyrosine 394, to phenylalanine diminished the enhancement of TCR-induced tyrosine protein phosphorylation by F505 p56lck. Such a finding is consistent with the previous demonstration that this site is required for full activation of p56lck by mutation of tyrosine 505. Strikingly, deletion of the noncatalytic Src homology domain 2, but not of the Src homology domain 3, markedly reduced the improvement of TCR-induced tyrosine protein phosphorylation by F505 Lck. Additional studies revealed that all the mutations tested, including deletion of the Src homology 3 region, abrogated the enhancement of antigen-triggered interleukin-2 production by F505 p56lck, thus implying more stringent requirements for augmentation of antigen responsiveness by F505 Lck. Finally, it was also observed that expression of F505 p56lck greatly increased TCR-induced tyrosine phosphorylation of phospholipase C-gamma 1, raising the possibility that phospholipase C-gamma 1 may be a substrate for p56lck in T lymphocytes. Our results indicate that p56lck regulates T-cell antigen receptor signalling through a complex process requiring multiple distinct structural domains of the protein.

scholarly journals Programmed cell death 1 forms negative costimulatory microclusters that directly inhibit T cell receptor signaling by recruiting phosphatase SHP2

2012 ◽  
Vol 209 (6) ◽  
pp. 1201-1217 ◽  
Author(s):  
Tadashi Yokosuka ◽  
Masako Takamatsu ◽  
Wakana Kobayashi-Imanishi ◽  
Akiko Hashimoto-Tane ◽  
Miyuki Azuma ◽  
...  
Keyword(s):  
Cell Death ◽  
T Cell ◽  
Programmed Cell Death ◽  
T Cell Activation ◽  
Cell Activation ◽  
Tyrosine Phosphatase ◽  
Cell Receptor ◽  
Programmed Cell Death 1

Programmed cell death 1 (PD-1) is a negative costimulatory receptor critical for the suppression of T cell activation in vitro and in vivo. Single cell imaging elucidated a molecular mechanism of PD-1–mediated suppression. PD-1 becomes clustered with T cell receptors (TCRs) upon binding to its ligand PD-L1 and is transiently associated with the phosphatase SHP2 (Src homology 2 domain–containing tyrosine phosphatase 2). These negative costimulatory microclusters induce the dephosphorylation of the proximal TCR signaling molecules. This results in the suppression of T cell activation and blockade of the TCR-induced stop signal. In addition to PD-1 clustering, PD-1–TCR colocalization within microclusters is required for efficient PD-1–mediated suppression. This inhibitory mechanism also functions in PD-1hi T cells generated in vivo and can be overridden by a neutralizing anti–PD-L1 antibody. Therefore, PD-1 microcluster formation is important for regulation of T cell activation.

scholarly journals 173 Expression of a membrane-tethered IL-15/IL-15 receptor fusion protein enhances the persistence of MSLN-targeted TRuC-T cells

2021 ◽  
Vol 9 (Suppl 3) ◽  
pp. A185-A185
Author(s):  
Michelle Fleury ◽  
Derrick McCarthy ◽  
Holly Horton ◽  
Courtney Anderson ◽  
Amy Watt ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Fusion Protein ◽  
T Cell Receptor ◽  
Cytokine Production ◽  
Cell Receptor ◽  
Great Promise ◽  
And Function

BackgroundAdoptive cell therapies have shown great promise in hematological malignancies but have yielded little progress in the context of solid tumors. We have developed T cell receptor fusion construct (TRuC®) T cells, which are equipped with an engineered T cell receptor that utilizes the full complement of TCR signaling subunits and recognizes tumor-associated antigens independent of HLA. In clinical trials, mesothelin (MSLN)-targeting TRuC-T cells (TC-210 or gavo-cel) have shown unprecedented results in patients suffering from advanced mesothelioma and ovarian cancer. To potentially increase the depth of response, we evaluated strategies that can promote intra-tumoral T cell persistence and function. Among the common ??-chain cytokines, IL-15 uniquely supports the differentiation and maintenance of memory T cell subsets by limiting terminal differentiation and conferring resistance to IL-2 mediated activation-induced cell death (AICD). In the studies described here, we evaluated the potential of IL-15 as an enhancement to TRuC-T cell phenotype, persistence and function against MSLN+ targets.MethodsPrimary human T cells were activated and transduced with a lentiviral vector encoding an anti-MSLN binder fused to CD3ε alone or co-expressed with a membrane-tethered IL-15rα/IL-15 fusion protein (IL-15fu). Transduced T cells were expanded for 9 days and characterized for expression of the TRuC, IL-15rα and memory phenotype before subjecting them to in vitro functional assays to evaluate cytotoxicity, cytokine production, and persistence. In vivo efficacy was evaluated in MHC class I/II deficient NSG mice bearing human mesothelioma xenografts.ResultsIn vitro, co-expression of the IL-15fu led to similar cytotoxicity and cytokine production as TC-210, but notably enhanced T-cell expansion and persistence upon repeated stimulation with MSLN+ cell lines. Furthermore, the IL-15fu-enhanced TRuC-T cells sustained a significantly higher TCF-1+ population and retained a stem-like phenotype following activation. Moreover, the IL-15fu-enhanced TRuCs demonstrated robust in vivo expansion and intra-tumoral accumulation as measured by ex vivo analysis of TRuC+ cells in the tumor and blood, with a preferential expansion of CD8+ T cells. Finally, IL-15fu-enhanced TRuC-T cells could be observed in the blood long after the tumors were cleared.ConclusionsThese pre-clinical studies suggest that the IL-15fu can synergize with TC-210 to increase the potency and durability of response in patients with MSLN+ tumors.Ethics ApprovalAll animal studies were approved by the respective Institutional Animal Care and Use Committees.

scholarly journals CCDC88B is a novel regulator of maturation and effector functions of T cells during pathological inflammation

2014 ◽  
Vol 211 (13) ◽  
pp. 2519-2535 ◽  
Author(s):  
James M. Kennedy ◽  
Nassima Fodil ◽  
Sabrina Torre ◽  
Silayuv E. Bongfen ◽  
Jean-Frédéric Olivier ◽  
...  
Keyword(s):  
T Cell ◽  
Cell Function ◽  
Coiled Coil ◽  
Cell Receptor ◽  
Pleiotropic Effect ◽  
Experimental Cerebral Malaria ◽  
A Genome ◽  
Important Regulator

We used a genome-wide screen in mutagenized mice to identify genes which inactivation protects against lethal neuroinflammation during experimental cerebral malaria (ECM). We identified an ECM-protective mutation in coiled-coil domain containing protein 88b (Ccdc88b), a poorly annotated gene that is found expressed specifically in spleen, bone marrow, lymph nodes, and thymus. The CCDC88B protein is abundantly expressed in immune cells, including both CD4+ and CD8+ T lymphocytes, and in myeloid cells, and loss of CCDC88B protein expression has pleiotropic effects on T lymphocyte functions, including impaired maturation in vivo, significantly reduced activation, reduced cell division as well as impaired cytokine production (IFN-γ and TNF) in response to T cell receptor engagement, or to nonspecific stimuli in vitro, and during the course of P. berghei infection in vivo. This identifies CCDC88B as a novel and important regulator of T cell function. The human CCDC88B gene maps to the 11q13 locus that is associated with susceptibility to several inflammatory and auto-immune disorders. Our findings strongly suggest that CCDC88B is the morbid gene underlying the pleiotropic effect of the 11q13 locus on inflammation.

Early Maturation of T-Cell Progenitors in the Absence of Glucocorticoids

Blood ◽  
1999 ◽  
Vol 94 (8) ◽  
pp. 2819-2826 ◽  
Author(s):  
Rosa Sacedón ◽  
Angeles Vicente ◽  
Alberto Varas ◽  
Eva Jiménez ◽  
Juan José Muñoz ◽  
...  
Keyword(s):  
Cell Differentiation ◽  
T Cell ◽  
Glucocorticoid Receptors ◽  
Fetal Liver ◽  
Cell Receptor ◽  
T Cell Differentiation ◽  
Pregnant Rats ◽  
Early Maturation

In the present work, we demonstrated that both fetal liver and thymic T-cell precursors express glucocorticoid receptors (GRs) indirectly suggesting a role for glucocorticoids (GCs) in the earliest events of T-cell differentiation. To evaluate this issue, we analyzed the thymic ontogeny in the progeny of adrenalectomized pregnant rats (Adx fetuses), an in vivo experimental model, which ensures the absence of circulating GCs until the establishment of the fetal hypothalamus-pituitary-adrenal (HPA) axis. In the absence of maternal GCs, T-cell development was significantly accelerated, the process being reversed by in vivo GC replacement. Mature single positive thymocytes (both CD4 and CD8) appeared in 16-day old fetal Adx thymus when in the control fetuses, most thymocytes still remained in the double-negative (DN) CD4−CD8− cell compartment. In addition, emigration of T-cell receptor (TcR)β positive cells to the spleen also occurred earlier in Adx fetuses than in control ones. In vitro recolonization of cultured deoxiguanosine-treated mouse fetal thymus lobes with 13-day-old fetal liver cell suspensions from both Adx and control fetuses demonstrated changes in the developmental capabilities of fetal liver T-cell precursors from embryos grown in the absence of GCs. Furthermore, a precocious lymphoid colonization of the thymic primordium from Adx fetuses was evidenced by ultrastructural analysis of both Adx and Sham early thymus. Both findings accounted for the accelerated T-cell differentiation observed in Adx fetuses. Together, these results support a role for GCs not only in the thymic cell death, but also in the early steps of T-cell differentiation.

Anatomic location and T-cell stimulatory functions of mouse dendritic cell subsets defined by CD4 and CD8 expression

Blood ◽  
2002 ◽  
Vol 99 (6) ◽  
pp. 2084-2093 ◽  
Author(s):  
Alexander D. McLellan ◽  
Michaela Kapp ◽  
Andreas Eggert ◽  
Christian Linden ◽  
Ursula Bommhardt ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cd8 T Cells ◽  
Antigen Expression ◽  
Cell Receptor ◽  
In Vitro Assays ◽  
Peptide Complexes ◽  
Marginal Zones

Abstract Mouse spleen contains CD4+, CD8α+, and CD4−/CD8α− dendritic cells (DCs) in a 2:1:1 ratio. An analysis of 70 surface and cytoplasmic antigens revealed several differences in antigen expression between the 3 subsets. Notably, the Birbeck granule–associated Langerin antigen, as well as CD103 (the mouse homologue of the rat DC marker OX62), were specifically expressed by the CD8α+ DC subset. All DC types were apparent in the T-cell areas as well as in the splenic marginal zones and showed similar migratory capacity in collagen lattices. The 3 DC subtypes stimulated allogeneic CD4+ T cells comparably. However, CD8α+ DCs were very weak stimulators of resting or activated allogeneic CD8+ T cells, even at high stimulator-to-responder ratios, although this defect could be overcome under optimal DC/T cell ratios and peptide concentrations using CD8+ F5 T-cell receptor (TCR)–transgenic T cells. CD8α− or CD8α+DCs presented alloantigens with the same efficiency for lysis by cytotoxic T lymphocytes (CTLs), and their turnover rate of class I–peptide complexes was similar, thus neither an inability to present, nor rapid loss of antigenic complexes from CD8α DCs was responsible for the low allostimulatory capacity of CD8α+ DCs in vitro. Surprisingly, both CD8α+ DCs and CD4−/CD8− DCs efficiently primed minor histocompatibility (H-Y male antigen) cytotoxicity following intravenous injection, whereas CD4+ DCs were weak inducers of CTLs. Thus, the inability of CD8α+ DCs to stimulate CD8+ T cells is limited to certain in vitro assays that must lack certain enhancing signals present during in vivo interaction between CD8α+ DCs and CD8+ T cells.

Signaling via LAT (linker for T-cell activation) and Syk/ZAP70 is required for ERK activation and NFAT transcriptional activation following CD2 stimulation

Blood ◽  
2000 ◽  
Vol 96 (6) ◽  
pp. 2181-2190 ◽  
Author(s):  
Maria Paola Martelli ◽  
Huamao Lin ◽  
Weiguo Zhang ◽  
Lawrence E. Samelson ◽  
Barbara E. Bierer
Keyword(s):  
T Cells ◽  
T Cell ◽  
T Cell Activation ◽  
Transcriptional Activation ◽  
Cell Activation ◽  
Interleukin 2 ◽  
Cell Receptor ◽  
Adapter Protein ◽  
Activated T Cells

Abstract Activation of T cells can be initiated through cell surface molecules in addition to the T-cell receptor-CD3 (TCR-CD3) complex. In human T cells, ligation of the CD2 molecule by mitogenic pairs of anti-CD2 monoclonal antibodies activates T cells via biochemical signaling pathways similar but not identical to those elicited on TCR engagement. This study describes a key role for the p36/38 membrane adapter protein linker for T cell activation (LAT) in CD2-mediated T-cell activation. Following ligation of CD2 on the surface of the Jurkat T-cell line and human purified T cells, LAT was tyrosine phosphorylated and shown to associate in vivo with a number of other tyrosine phosphorylated proteins including PLCγ-1, Grb-2, and SLP-76. Using Jurkat cell lines deficient in ZAP70/Syk (P116) or LAT (ANJ3) expression, CD2-dependent PLCγ-1 and SLP-76 tyrosine phosphorylation required expression both of ZAP70 or Syk and of LAT. As predicted, the absence of either LAT or ZAP70/Syk kinases correlated with a defect in the induction of nuclear factor of activated T cells (NFAT) transcriptional activity, activation of the interleukin-2 promoter, and ERK phosphorylation following CD2 stimulation. These data suggest that LAT is an adapter protein important for the regulation of CD2-mediated T-cell activation.

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