scholarly journals Centrioles control the capacity, but not the specificity, of cytotoxic T cell killing

2020 ◽  
Vol 117 (8) ◽  
pp. 4310-4319 ◽  
Author(s):  
Fella Tamzalit ◽  
Diana Tran ◽  
Weiyang Jin ◽  
Vitaly Boyko ◽  
Hisham Bazzi ◽  
...  
Keyword(s):  
Cytotoxic T Lymphocytes ◽  
Synapse Formation ◽  
Immunological Synapse ◽  
Cytotoxic T Cell ◽  
Target Cells ◽  
Synaptic Membrane ◽  
Structural Components ◽  
Actin Remodeling ◽  
The Core ◽  
Lytic Granule

Immunological synapse formation between cytotoxic T lymphocytes (CTLs) and the target cells they aim to destroy is accompanied by reorientation of the CTL centrosome to a position beneath the synaptic membrane. Centrosome polarization is thought to enhance the potency and specificity of killing by driving lytic granule fusion at the synapse and thereby the release of perforin and granzymes toward the target cell. To test this model, we employed a genetic strategy to delete centrioles, the core structural components of the centrosome. Centriole deletion altered microtubule architecture as expected but surprisingly had no effect on lytic granule polarization and directional secretion. Nevertheless, CTLs lacking centrioles did display substantially reduced killing potential, which was associated with defects in both lytic granule biogenesis and synaptic actin remodeling. These results reveal an unexpected role for the intact centrosome in controlling the capacity but not the specificity of cytotoxic killing.

scholarly journals Centrioles control the capacity, but not the specificity, of cytotoxic T cell killing

2019 ◽  
Author(s):  
Fella Tamzalit ◽  
Diana Tran ◽  
Weiyang Jin ◽  
Vitaly Boyko ◽  
Hisham Bazzi ◽  
...  
Keyword(s):  
Cytotoxic T Lymphocytes ◽  
Synapse Formation ◽  
Immunological Synapse ◽  
Cytotoxic T Cell ◽  
Target Cells ◽  
Synaptic Membrane ◽  
Structural Components ◽  
Actin Remodeling ◽  
The Core ◽  
Lytic Granule

Immunological synapse formation between cytotoxic T lymphocytes (CTLs) and the target cells they aim to destroy is accompanied by reorientation of the CTL centrosome to a position beneath the synaptic membrane. Centrosome polarization is thought to enhance the potency and specificity of killing by driving lytic granule fusion at the synapse and thereby the release of perforin and granzymes toward the target cell. To test this model, we employed a genetic strategy to delete centrioles, the core structural components of the centrosome. Centriole deletion altered microtubule architecture, as expected, but surprisingly had no effect on lytic granule polarization and directional secretion. Nevertheless, CTLs lacking centrioles did display substantially reduced killing potential, which was associated with defects in both lytic granule biogenesis and synaptic actin remodeling. These results reveal an unexpected role for the intact centrosome in controlling the capacity, but not the specificity, of cytotoxic killing.

scholarly journals Extracellular Cystatin F Is Internalised by Cytotoxic T Lymphocytes and Decreases Their Cytotoxicity

Cancers ◽  
2020 ◽  
Vol 12 (12) ◽  
pp. 3660
Author(s):  
Mateja Prunk ◽  
Milica Perišić Nanut ◽  
Tanja Jakoš ◽  
Jerica Sabotič ◽  
Urban Švajger ◽  
...  
Keyword(s):  
T Lymphocytes ◽  
Cytotoxic T Lymphocytes ◽  
Cathepsin L ◽  
Killer Cell ◽  
Full Length ◽  
Cytotoxic T Cell ◽  
Target Cells ◽  
Natural Killer Cell Cytotoxicity ◽  
Cathepsin C ◽  
Cysteine Cathepsins

Cystatin F is a protein inhibitor of cysteine cathepsins, peptidases involved in the activation of the effector molecules of the perforin/granzyme pathway. Cystatin F was previously shown to regulate natural killer cell cytotoxicity. Here, we show that extracellular cystatin F has a role in regulating the killing efficiency of cytotoxic T lymphocytes (CTLs). Extracellular cystatin F was internalised into TALL-104 cells, a cytotoxic T cell line, and decreased their cathepsin C and H activity. Correspondingly, granzyme A and B activity was also decreased and, most importantly, the killing efficiency of TALL-104 cells as well as primary human CTLs was reduced. The N-terminally truncated form of cystatin F, which can directly inhibit cathepsin C (unlike the full-length form), was more effective than the full-length inhibitor. Furthermore, cystatin F decreased cathepsin L activity, which, however, did not affect perforin processing. Cystatin F derived from K-562 target cells could also decrease the cytotoxicity of TALL-104 cells. These results clearly show that, by inhibiting cysteine cathepsin proteolytic activity, extracellular cystatin F can decrease the cytotoxicity of CTLs and thus compromise their function.

scholarly journals The control of specificity of cytotoxic T lymphocytes by the major histocompatibility complex (AG-B) in rats and identification of a new alloantigen system showing no AG-B restriction.

1977 ◽  
Vol 146 (6) ◽  
pp. 1773-1790 ◽  
Author(s):  
A Marshak ◽  
P C Doherty ◽  
D B Wilson
Keyword(s):  
Major Histocompatibility Complex ◽  
T Lymphocytes ◽  
Cytotoxic T Lymphocytes ◽  
Third Party ◽  
Cytotoxic T Cell ◽  
Target Cells ◽  
Cytotoxic Lymphocytes ◽  
Major Histocompatibility ◽  
Histocompatibility Complex ◽  
Ct Antigens

The regulatory influence of the rat major histocompatibility complex (MHC) (Ag-B complex) on the specificity of cytotoxic T lymphocytes was investigated. It was shown that the effector cells were specific for the original Ag-B phenotype in rat systems in which the responder and stimulator cell populations were unquestionably MHC identical but expressed different minor alloantigens of viral antigens. However, combined in vivo immunization and restimulation in culture of lymphocytes from rat strains previously thought to be MHC compatible resulted in the generation of cytotoxic T lymphocytes which effectively lyse not only target cells from the specific stimulating strains but also, to varying degrees, target cells from third party strains regardless of their Ag-B haplotypes. Genetic analysis indicates that expression of these cytotoxic T-cell-defined ("CT") antigens, found on both T and B lymphocytes, detectable thus far only with cytotoxic lymphocytes, is controlled by a single locus which segregates in backcross populations with the rat MHC. Discrepancies between the nature of CT antigens of the rat Ag-B and I-region specificities of the mouse H-2 are discussed.

Targeting of «T» Lymphocytes against Human Hepatoma Cells by a Bispecific Monoclonal Antibody: Role of Different Lymphocyte Subsets

1992 ◽  
Vol 78 (2) ◽  
pp. 79-86 ◽  
Author(s):  
Qi Chen ◽  
Peinan Sun ◽  
Ignazia Prigione ◽  
Hong Xie ◽  
Silvano Ferrini
Keyword(s):  
T Lymphocytes ◽  
Cytotoxic T Lymphocytes ◽  
Hepatoma Cells ◽  
Peripheral Blood Lymphocytes ◽  
Cytolytic Activity ◽  
Cytotoxic T Cell ◽  
Target Cells ◽  
Human Hepatoma ◽  
Human Hepatoma Cells

In an attempt to construct bispecific monoclonal antibodies (bimAbs) able to target cytotoxic T lymphocytes against human hepatoma cells, an HGPRT-deficient mutant of the Hepama-6 hybridoma, which produces an antihuman-hepatoma mAb, was directly fused with splenocytes from Balb/C mice immunized by a polyclonal cytotoxic T-cell line. Hybrid hybridomas were selected in HAT medium, and their supernatants were directly screened for the ability to induce IL-2-cultured cytotoxic T lymphocytes to kill hepatoma cells in a 51Cr-release assay. The selected hybrid hybridoma, termed DQ-33, secretes a bimAb, which reacts with a CD3-associated determinant. When resting peripheral-blood lymphocytes were used as effector cells, virtually no cytolytic activity could be induced by DQ-33, whereas phytohemagglutinin-activated lymphocytes that had been expanded in vitro in IL-2-containing medium could be efficiently targeted against hepatoma cells. Targeting by DQ-33 bimAb was analyzed on different subsets of IL-2-cultured lymphocytes. It was evident that CD+4–8+ TCRα/β+ and CD3+4–8-TCRγ/δ+ lymphocytes were efficiently induced by bimAb to lyse human hepatoma cells, whereas no induction of cytolysis could be observed when CD3 + 4+8-TCRα/β+ cells were used as effectors. DQ-33 bimAb was also able to induce lymphokine secretion (IL-2, GM-CSF and TNF-α) by all the different subsets of lymphocytes analyzed in the presence of target cells expressing the relevant antigen, independent of the expression of cytolytic activity.

scholarly journals Rab27a Is Required for Regulated Secretion in Cytotoxic T Lymphocytes

2001 ◽  
Vol 152 (4) ◽  
pp. 825-834 ◽  
Author(s):  
Jane C. Stinchcombe ◽  
Duarte C. Barral ◽  
Emilie H. Mules ◽  
Sarah Booth ◽  
Alistair N. Hume ◽  
...  
Keyword(s):  
T Lymphocytes ◽  
Cytotoxic T Lymphocytes ◽  
Immunological Synapse ◽  
Target Cells ◽  
Rab Proteins ◽  
Loss Of Function ◽  
Α Subunit ◽  
Lytic Granules ◽  
Granzyme A ◽  
Geranylgeranyl Transferase

Rab27a activity is affected in several mouse models of human disease including Griscelli (ashen mice) and Hermansky-Pudlak (gunmetal mice) syndromes. A loss of function mutation occurs in the Rab27a gene in ashen (ash), whereas in gunmetal (gm) Rab27a dysfunction is secondary to a mutation in the α subunit of Rab geranylgeranyl transferase, an enzyme required for prenylation and activation of Rabs. We show here that Rab27a is normally expressed in cytotoxic T lymphocytes (CTLs), but absent in ashen homozygotes (ash/ash). Cytotoxicity and secretion assays show that ash/ash CTLs are unable to kill target cells or to secrete granzyme A and hexosaminidase. By immunofluorescence and electron microscopy, we show polarization but no membrane docking of ash/ash lytic granules at the immunological synapse. In gunmetal CTLs, we show underprenylation and redistribution of Rab27a to the cytosol, implying reduced activity. Gunmetal CTLs show a reduced ability to kill target cells but retain the ability to secrete hexosaminidase and granzyme A. However, only some of the granules polarize to the immunological synapse, and many remain dispersed around the periphery of the CTLs. These results demonstrate that Rab27a is required in a final secretory step and that other Rab proteins also affected in gunmetal are likely to be involved in polarization of the granules to the immunological synapse.

scholarly journals Peptide-independent Recognition by Alloreactive Cytotoxic T Lymphocytes (CTL)

1997 ◽  
Vol 185 (6) ◽  
pp. 1023-1034 ◽  
Author(s):  
Pamela A. Smith ◽  
Anders Brunmark ◽  
Michael R. Jackson ◽  
Terry A. Potter
Keyword(s):  
T Cells ◽  
T Lymphocytes ◽  
Cytotoxic T Lymphocytes ◽  
Skin Graft ◽  
Antigen Processing ◽  
Cytotoxic T Cell ◽  
Target Cells ◽  
Mhc Molecules ◽  
Alloreactive T Cells

We have isolated several H-2Kb–alloreactive cytotoxic T cell clones and analyzed their reactivity for several forms of H-2Kb. These cytotoxic T lymphocytes (CTL) were elicited by priming with a skin graft followed by in vitro stimulation using stimulator cells that express an H-2Kb molecule unable to bind CD8. In contrast to most alloreactive T cells, these CTL were able to recognize H-2Kb on the surface of the antigen processing defective cell lines RMA-S and T2. Furthermore, this reactivity was not increased by the addition of an extract containing peptides from C57BL/6 (H-2b) spleen cells, nor was the reactivity decreased by treating the target cells with acid to remove peptides bound to MHC molecules. The CTL were also capable of recognizing targets expressing the mutant H-2Kbm8 molecule. These findings suggested that the clones recognized determinants on H-2Kb that were independent of peptide. Further evidence for this hypothesis was provided by experiments in which H-2Kb produced in Drosophila melanogaster cells and immobilized on the surface of a tissue culture plate was able to stimulate hybridomas derived from these alloreactive T cells. Precursor frequency analysis demonstrated that skin graft priming, whether with skin expressing the wild-type or the mutant H-2Kb molecule, is a strong stimulus to elicit peptide-independent CTL. Moreover, reconstitution experiments demonstrated that the peptide-independent CTL clones were capable of mediating rapid and complete rejection of H-2–incompatible skin grafts. These findings provide evidence that not all allorecognition is peptide dependent.

scholarly journals Cortical actin recovery at the immunological synapse leads to termination of lytic granule secretion in cytotoxic T lymphocytes

2017 ◽  
Vol 114 (32) ◽  
pp. E6585-E6594 ◽  
Author(s):  
Alex T. Ritter ◽  
Senta M. Kapnick ◽  
Sricharan Murugesan ◽  
Pamela L. Schwartzberg ◽  
Gillian M. Griffiths ◽  
...  
Keyword(s):  
T Lymphocytes ◽  
Cytotoxic T Lymphocytes ◽  
Immunological Synapse ◽  
Cortical Actin ◽  
Immune Synapse ◽  
Superresolution Microscopy ◽  
Lytic Granules ◽  
Infected Cells ◽  
Granule Secretion ◽  
Lytic Granule

CD8+ cytotoxic T lymphocytes (CTLs) eliminate virally infected cells through directed secretion of specialized lytic granules. Because a single CTL can kill multiple targets, degranulation must be tightly regulated. However, how CTLs regulate the termination of granule secretion remains unclear. Previous work demonstrated that centralized actin reduction at the immune synapse precedes degranulation. Using a combination of live confocal, total internal reflection fluorescence, and superresolution microscopy, we now show that, after granule fusion, actin recovers at the synapse and no further secretion is observed. Depolymerization of actin led to resumed granule secretion, suggesting that recovered actin acts as a barrier preventing sustained degranulation. Furthermore, RAB27a-deficient CTLs, which do not secrete cytotoxic granules, failed to recover actin at the synapse, suggesting that RAB27a-mediated granule secretion is required for actin recovery. Finally, we show that both actin clearance and recovery correlated with synaptic phosphatidylinositol 4,5-bisphosphate (PIP2) and that alterations in PIP2 at the immunological synapse regulate cortical actin in CTLs, providing a potential mechanism through which CTLs control cortical actin density. Our work provides insight into actin-related mechanisms regulating CTL secretion that may facilitate serial killing during immune responses.

scholarly journals Centrosome docking at the immunological synapse is controlled by Lck signaling

2011 ◽  
Vol 192 (4) ◽  
pp. 663-674 ◽  
Author(s):  
Andy Tsun ◽  
Ihjaaz Qureshi ◽  
Jane C. Stinchcombe ◽  
Misty R. Jenkins ◽  
Maike de la Roche ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
T Cell ◽  
Tyrosine Kinase ◽  
T Cell Receptor ◽  
Cytotoxic T Lymphocytes ◽  
Immunological Synapse ◽  
Cell Receptor ◽  
Target Cells ◽  
Lytic Granules ◽  
Distal Side

Docking of the centrosome at the plasma membrane directs lytic granules to the immunological synapse. To identify signals controlling centrosome docking at the synapse, we have studied cytotoxic T lymphocytes (CTLs) in which expression of the T cell receptor–activated tyrosine kinase Lck is ablated. In the absence of Lck, the centrosome is able to translocate around the nucleus toward the immunological synapse but is unable to dock at the plasma membrane. Lytic granules fail to polarize and release their contents, and target cells are not killed. In CTLs deficient in both Lck and the related tyrosine kinase Fyn, centrosome translocation is impaired, and the centrosome remains on the distal side of the nucleus relative to the synapse. These results show that repositioning of the centrosome in CTLs involves at least two distinct steps, with Lck signaling required for the centrosome to dock at the plasma membrane.

scholarly journals Annular PIP3 accumulation controls actin architecture and modulates cytotoxicity at the immunological synapse

2013 ◽  
Vol 210 (12) ◽  
pp. 2721-2737 ◽  
Author(s):  
Audrey Le Floc’h ◽  
Yoshihiko Tanaka ◽  
Niels S. Bantilan ◽  
Guillaume Voisinne ◽  
Grégoire Altan-Bonnet ◽  
...  
Keyword(s):  
T Cell ◽  
Actin Polymerization ◽  
Immunological Synapse ◽  
Cell Receptor ◽  
Synaptic Membrane ◽  
Filamentous Actin ◽  
Actin Ring ◽  
Actin Remodeling ◽  
Peripheral Ring ◽  
Rho Family

The immunological synapse formed by a T lymphocyte on the surface of a target cell contains a peripheral ring of filamentous actin (F-actin) that promotes adhesion and facilitates the directional secretion of cytokines and cytolytic factors. We show that growth and maintenance of this F-actin ring is dictated by the annular accumulation of phosphatidylinositol trisphosphate (PIP3) in the synaptic membrane. PIP3 functions in this context by recruiting the exchange factor Dock2 to the periphery of the synapse, where it drives actin polymerization through the Rho-family GTPase Rac. We also show that synaptic PIP3 is generated by class IA phosphoinositide 3-kinases that associate with T cell receptor microclusters and are activated by the GTPase Ras. Perturbations that inhibit or promote PIP3-dependent F-actin remodeling dramatically affect T cell cytotoxicity, demonstrating the functional importance of this pathway. These results reveal how T cells use lipid-based signaling to control synaptic architecture and modulate effector responses.

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