The mechanism of T cell mediated cytotoxicity V. Morphological studies by electron microscopy

1977 ◽  
Vol 198 (1132) ◽  
pp. 315-323 ◽  
Keyword(s):  
T Cells ◽  
Cell Death ◽  
T Cell ◽  
Target Cell ◽  
Plasma Membranes ◽  
Ultrastructural Changes ◽  
Early Event ◽  
Target Cells ◽  
Cell Functions ◽  
Morphological Studies

The ultrastructural changes occurring when T cells specifically immune to antigens interact with P815 mastocytoma cells and EL4 lymphoma cells are described and related to changes previously observed by timelapse cinematography (Sanderson 1976 b ). In confirmation of work by others, pale T cells can clearly be in­criminated as cytotoxic cells. Dark T cells also form contacts with target cells, and tend to project lamellipodia over the surface of the target cell. The possibility is discussed that these represent a subset of non-cytotoxic, antigen-reactive T cells involved in other T cell functions. T cells form two types of contact: relatively small point contacts, and large areas where the two plasma membranes are in close apposition. No structures resembling specialized junctions or membrane fusions were observed in areas of contact between T cells and target cells. Close contact between pale T cells and target cells is more regular than contact with dark T cells. Many contacts were seen between morphologically normal target cells and pale T cells, and these were thought to occur in the phase between contact and target cell death. Some of these pale T cells in contact show projections towards the centre of the target cell which invaginate the cell membrane, but do not penetrate it. Remarkable T cell projections were also seen which had penetrated deeply through the membrane of one target cell. These projections appeared to have disrupted the mem­brane and had penetrated into apparently intact cytoplasm, suggesting that this penetration may be an early event in the lytic mechanism. The possibility that this phenomenon is the cause of cell death is discussed. Changes corresponding to the phase of zeiosis of the target cell are described. These commence with the formation of surface blebs, accompanied by a general mis-shapening of the cell outline and followed by vacuolation and loss of cytoplasmic organelles. Breakdown of the nucleus appears to be a later, post mortem event.

New CFSE-based assay to determine susceptibility to lysis by cytotoxic T cells of leukemic precursor cells within a heterogeneous target cell population

Blood ◽  
2004 ◽  
Vol 103 (7) ◽  
pp. 2677-2682 ◽  
Author(s):  
Inge Jedema ◽  
Nicole M. van der Werff ◽  
Renée M. Y. Barge ◽  
Roel Willemze ◽  
J. H. Frederik Falkenburg
Keyword(s):  
T Cells ◽  
Cell Death ◽  
T Cell ◽  
Cell Population ◽  
Target Cell ◽  
Precursor Cells ◽  
Leukemic Cells ◽  
Cell Populations ◽  
Target Cells ◽  
Specific Cell

Abstract For the clinical evaluation of the efficacy of cellular immunotherapy it is necessary to analyze the effector functions of T cells against primary leukemic target cell populations which are usually considerably heterogeneous caused by differential maturation stages of the leukemic cells. An appropriate assay should not only allow the quantitative analysis of rapid cell death induction as measured by the conventional 51Cr release assay but also of the more slowly executing pathways of T-cell-induced apoptosis occurring within days instead of hours which cannot be measured using this method. Furthermore, it should dissect the differential susceptibility to T-cell-induced cell death of various target cell subpopulations and characterize the malignant precursor cells capable of producing malignant progeny. To fulfill these requirements we developed a new assay based on carboxyfluorescein diacetate succinimidyl ester (CFSE) labeling of the target cell population combined with antibody staining of specific cell populations and addition of fluorescent microbeads to quantitatively monitor target cell death occurring within a longer time frame up to at least 5 days. This new assay facilitates the analysis of differential recognition of distinct cell types within a heterogeneous target cell population and allows simultaneously evaluation of the proliferative status of surviving target cells in response to relevant cytokines. (Blood. 2004;103: 2677-2682)

scholarly journals Fas ligand mediates activation-induced cell death in human T lymphocytes.

1995 ◽  
Vol 181 (1) ◽  
pp. 71-77 ◽  
Author(s):  
M R Alderson ◽  
T W Tough ◽  
T Davis-Smith ◽  
S Braddy ◽  
B Falk ◽  
...  
Keyword(s):  
T Cells ◽  
Cell Death ◽  
T Cell ◽  
Fas Ligand ◽  
Significant Proportion ◽  
Cell Receptor ◽  
Target Cells ◽  
Activated T Cells ◽  
Activation Induced Cell Death ◽  
Fas L

A significant proportion of previously activated human T cells undergo apoptosis when triggered through the CD3/T cell receptor complex, a process termed activation-induced cell death (AICD). Ligation of Fas on activated T cells by either Fas antibodies or recombinant human Fas-ligand (Fas-L) also results in cytolysis. We demonstrate that these two pathways of apoptosis are causally related. Stimulation of previously activated T cells resulted in the expression of Fas-L mRNA and lysis of Fas-positive target cells. Fas-L antagonists inhibited AICD of T cell clones and staphylococcus enterotoxin B (SEB)-specific T cell lines. The data indicate AICD in previously stimulated T cells is mediated by Fas/Fas-L interactions.

scholarly journals Lenalidomide Enhances CAR-T Cell Activity Against Solid Tumor Cells

2020 ◽  
Vol 29 ◽  
pp. 096368972092082 ◽  
Author(s):  
Zhixiong Wang ◽  
Guomin Zhou ◽  
Na Risu ◽  
Jiayu Fu ◽  
Yan Zou ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Line ◽  
Cytokine Secretion ◽  
Target Cells ◽  
Functional Assay ◽  
Cell Functions ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T

Chimeric antigen receptor (CAR) T-cell immunotherapy still faces many challenges in the treatment of solid tumors, one of which is T-cell dysfunction or exhaustion. Immunomodulator lenalidomide may improve CAR T-cell function. In this study, the effects of lenalidomide on CAR T-cell functions (cytotoxicity, cytokine secretion, and cell proliferation) were investigated. Two different CAR T cells (CD133-specific CAR and HER2-specific CAR) were prepared, and the corresponding target cells including human glioma cell line U251 CD133-OE that overexpress CD133 and human breast cancer cell line MDA-MB-453 were used for functional assay. We found that lenalidomide promoted the killing of U251 CD133-OE by CD133-CAR T cells, the cytokine secretion, and the proliferation of CD133-CAR T cells. Lenalidomide also enhanced the cytotoxicity against MDA-MB-453 and the cytokine secretion of HER2-CAR T cells but did not affect their proliferation significantly. Furthermore, lenalidomide may regulate the function of CAR T cells by inducing the degradation of transcription factors Ikaros and Aiolos.

Fas-based d1OS-mediated cytotoxicity requires macromolecular synthesis for effector cell activation but not for target cell death

1994 ◽  
Vol 345 (1313) ◽  
pp. 303-309 ◽  
Keyword(s):  
Cell Death ◽  
T Cell ◽  
Cell Surface ◽  
Target Cell ◽  
Cell Activation ◽  
Effector Cell ◽  
Apoptotic Cell ◽  
Target Cells ◽  
Macromolecular Synthesis ◽  
Effector Cells

Two main mechanisms seem at play in T cell-mediated cytotoxicity, a process in which target cell death often follows an apoptotic cell death pattern. One of these involves Fas at the target cell surface and a Fas ligand at the effector cell surface. This allowed us to reinvestigate the long-standing question of macromolecular synthesis requirement in T cell-mediated cytotoxicity, using the dlOS model cell line which is cytotoxic apparently only via the Fas molecularly defined mechanism. We showed, first, that induction of cytotoxic activity of effector cells, obtained by preincubating these effector cells with a phorbol ester and a calcium ionophore, could be inhibited by macromolecular synthesis inhibitors (cycloheximide, actinomycin D, DRB). We then investigated whether macromolecular synthesis was required, when effector and target cells were mixed, to obtain target cell death. Preincubating already activated effector cells for 30 min with macromolecular synthesis inhibitors, then adding target cells and performing the 51 Cr release cytotoxicity test in the presence of these inhibitors, did not significantly decrease subsequent target cell death, indicating that already activated effector cells could kill without further requirement for macromolecular synthesis. In addition, target cell preincubation for up to 3 h in the presence of one of these inhibitors did not decrease cell death. The high sensitivity of mouse thymocytes to this type of cytotoxicity enabled us to devise the following experiment. As previously shown by others, thymocyte death induced by dexamethasone (DEX) could be blocked by coincubation with cycloheximide (CHX). Such DEX-treated CHX-rescued thymocytes, the survival of which was an internal control of efficiency of protein synthesis inhibition, were then subjected to effector cells in the presence of CHX, and were shown to die. Thus, there is no requirement for macromolecular synthesis at the target cell level in this variety of apoptotic cell death. Altogether, in this defined mechanism of T cell-mediated cytotoxicity, macromolecular synthesis is required for dlOS effector cell activation, but not for lysis by already activated effector cells nor for target cell death.

Activation of T Cells by Bispecific Single-Chain Construct MT103 (MEDI-538) Is Strictly Target Cell-Dependent.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 3889-3889
Author(s):  
Klaus Brischwein ◽  
Scott A. Hammond ◽  
Larissa Parr ◽  
Schlereth Bernd ◽  
Mathias Locher ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
T Cell Activation ◽  
Target Cell ◽  
Cell Activation ◽  
Cell Lysis ◽  
Target Cells ◽  
Single Chain ◽  
Activation Markers ◽  
Human T Cells

Abstract Background: Bispecific antibodies have been extensively studied in vitro and in vivo for their use in redirected tumor cell lysis. A particular challenge of bispecific antibody constructs recognizing the CD3 signaling complex is to achieve a controlled polyclonal activation of T-cells that, ideally, is entirely dependent on the presence of target cells. If this is not the case, systemic production of inflammatory cytokines and secondary endothelial reactions may occur as side effects, as are observed with the murine anti-human CD3e antibody OKT-3 (muromab, Orthoclone®). Here we present evidence that MT103 (or MEDI-538), a bispecific single chain antibody of the BiTE class that targets CD19 and CD3, induces T-cell activation exclusively in the presence of target cells. Material and methods: Peripheral blood mononuclear cells from healthy donors were prepared by Ficoll density centrifugation. PBMC were incubated for 24 hours with MT103 in presence or absence of specific target cells. Target cell lysis was determined by measurement of adenylate kinase activity released from lysed cells. De novo expression of activation markers CD69 and CD25 on T-cells was assessed by flow cytometry using directly conjugated monoclonal antibodies, and the concentration of cytokines in the supernatant was determined by a commercial FACS-based bead array. Results: MT103 was analyzed for conditional T-cell activation. In the presence of target-expressing cell lines, low picomolar concentrations of MT103 were sufficient to stimulate a high percentage of peripheral human T-cells to express cytokines and surface activation markers, to enter into the cell cycle and to induce redirected lysis of target cells. However, in the absence of target cells, the BiTE molecules no longer detectably activated human T-cells even at concentrations exceeding the ED50 for redirected lysis and conditional T-cell activation by more than five orders of magnitude. Conclusion: Our data show that T-cell activation by MT103 is highly conditional in that it is strictly dependent on the presence.

scholarly journals Regulation of T Cells in Cancer by Nitric Oxide

Cells ◽  
2021 ◽  
Vol 10 (10) ◽  
pp. 2655
Author(s):  
Inesa Navasardyan ◽  
Benjamin Bonavida
Keyword(s):  
Immune Response ◽  
T Cells ◽  
T Cell ◽  
Cd8 T Cells ◽  
Suppressor Cells ◽  
T Cell Response ◽  
T Cell Subsets ◽  
Target Cells ◽  
Effector Cells ◽  
Cell Functions

The T cell-mediated immune response is primarily involved in the fight against infectious diseases and cancer and its underlying mechanisms are complex. The anti-tumor T cell response is regulated by various T cell subsets and other cells and tissues in the tumor microenvironment (TME). Various mechanisms are involved in the regulation of these various effector cells. One mechanism is the iNOS/.NO that has been reported to be intimately involved in the regulation and differentiation of the various cells that regulate the anti-tumor CD8 T cells. Both endogenous and exogenous .NO are implicated in this regulation. Importantly, the exposure of T cells to .NO had different effects on the immune response, depending on the .NO concentration and time of exposure. For instance, iNOS in T cells regulates activation-induced cell death and inhibits Treg induction. Effector CD8 T cells exposed to .NO result in the upregulation of death receptors and enhance their anti-tumor cytotoxic activity. .NO-Tregs suppress CD4 Th17 cells and their differentiation. Myeloid-derived suppressor cells (MDSCs) expressing iNOS inhibit T cell functions via .NO and inhibit anti-tumor CD8 T cells. Therefore, both .NO donors and .NO inhibitors are potential therapeutics tailored to specific target cells that regulate the T cell effector anti-tumor response.

scholarly journals CRISPR/Cas9-mediated TGFβRII disruption enhances anti-tumor efficacy of human chimeric antigen receptor T cells in vitro

2021 ◽  
Vol 19 (1) ◽  
Author(s):  
Khadijeh Alishah ◽  
Matthias Birtel ◽  
Elham Masoumi ◽  
Leila Jafarzadeh ◽  
Hamid Reza Mirzaee ◽  
...  
Keyword(s):  
T Cells ◽  
Immune System ◽  
T Cell ◽  
Solid Tumors ◽  
Target Cells ◽  
Cell Functions ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T

Abstract Background CAR T-cell therapy has been recently unveiled as one of the most promising cancer therapies in hematological malignancies. However, solid tumors mount a profound line of defense to escape immunosurveillance by CAR T-cells. Among them, cytokines with an inhibitory impact on the immune system such as IL-10 and TGFβ are of great importance: TGFβ is a pleiotropic cytokine, which potently suppresses the immune system and is secreted by a couple of TME resident and tumor cells. Methods In this study, we hypothesized that knocking out the TGFβ receptor II gene, could improve CAR T-cell functions in vitro and in vivo. Hereby, we used the CRISPR/Cas9 system, to knockout the TGFβRII gene in T-cells and could monitor the efficient gene knock out by genome analysis techniques. Next, Mesothelin or Claudin 6 specific CAR constructs were overexpressed via IVT-RNA electroporation or retroviral transduction and the poly-functionality of these TGFβRII KO CAR T-cells in terms of proliferation, cytokine secretion and cytotoxicity were assessed and compared with parental CAR T-cells. Results Our experiments demonstrated that TGFβRII KO CAR T-cells fully retained their capabilities in killing tumor antigen positive target cells and more intriguingly, could resist the anti-proliferative effect of exogenous TGFβ in vitro outperforming wild type CAR T-cells. Noteworthy, no antigen or growth factor-independent proliferation of these TGFβRII KO CAR T-cells has been recorded. TGFβRII KO CAR T-cells also resisted the suppressive effect of induced regulatory T-cells in vitro to a larger extent. Repetitive antigen stimulation demonstrated that these TGFβRII KO CAR T-cells will experience less activation induced exhaustion in comparison to the WT counterpart. Conclusion The TGFβRII KO approach may become an indispensable tool in immunotherapy of solid tumors, as it may surmount one of the key negative regulatory signaling pathways in T-cells.

Resistance of Quiescent Cells to T Cell Attack Determines Both the Efficacy and Specificity of Adoptive T Cell Therapy.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 3161-3161
Author(s):  
I. Jedema ◽  
C.A.M. van Bergen ◽  
M.G.D. Kester ◽  
R. Willemze ◽  
J.H. Frederik Falkenburg
Keyword(s):  
T Cells ◽  
T Cell ◽  
B Cells ◽  
Immune Responses ◽  
Target Cell ◽  
Cytotoxic T Cells ◽  
Precursor Cells ◽  
Adoptive T Cell Therapy ◽  
Target Cells ◽  
T Cell Therapy

Abstract Although profound anti-leukemic immune responses can be induced with donor lymphocyte infusions in patients with relapsed or persistent leukemia after allogeneic stem cell transplantation, (late) relapses of the same disease develop regularly even in patients initially entering a complete remission. This suggests that a subpopulation of leukemic (precursor) cells with ultimate self-renewal capacity is capable of resisting T cell attack. We hypothesized that quiescent leukemic precursor cells can evade anti-leukemic therapy by their capacity to survive and persist in the presence of competent cytotoxic T cells. In addition, selectivity of cytotoxic T cells (CTLs) for target cells in active cell cycle in general may also explain why powerful immune responses directed against antigens that are broadly expressed on all tissues of the recipient, like the male-specific HY-antigens, do not necessarily result in severe damage to all tissues of the recipient. Therefore, we determined the efficacy of high affinity CTL clones directed against allo-HLA or minor histocompatibility antigens to kill normal and leukemic hematopoietic cells in dormancy and in active cell cycle, comprising normal and leukemic CD34+ precursor cells, normal B cells, T cells and monocytes, and activated B cells (EBV-LCL) and activated T cells (PHA blasts). Using a CFSE-based cytotoxicity assay allowing the analysis of susceptibility to lysis of specific cell types within a heterogeneous target cell population, we found that all activated target cells were very efficiently lysed, resulting in 60–90% lysis already after 4 hours of exposure to the CTL clones (E/T ratios 1/1–5/1). In contrast, target cells in relative dormancy including the non-proliferating CD34+ CML stem cell fraction, unmanipulated CD34 progenitor cells, and resting T and B cells were protected from CTL-induced cell death (0–20% lysis). Since normal expression of adhesion and HLA class I molecules was shown on these dormant cells, we investigated whether decreased avidity of the T cell/target cell interaction was underlying the poor susceptibility. Therefore, we artificially enhanced the avidity by exogenous loading of the target cells with saturating concentrations of the relevant peptide. This was sufficient to restore the sensitivity to levels comparable to activated target cells, suggesting that decreased avidity of the interaction between high affinity CTL and resting target cells plays a role in the resistance phenomenon. However, even after restoration of the high avidity interaction, a small population of (leukemic) target cells (0,1–10% of the total cell population) was capable of residing, suggesting that additional factors like resistance of quiescent target cells to one or more of the T cell effector mechanisms are involved. To analyze the influence of the sensitivity to T cell lysis of specific target cell types on the specificity of adoptive T cell therapy, we used non-hematopoietic target cells like mesenchymal stem cells and biliary epithelium cells as target cells. Alloreactive T cells showed also diminished capacity to lyse these target cells (10–20% lysis). The addition of inflammatory cytokines like TNF and interferons slightly increased the recognition. In conclusion, under steady state conditions potent allo immune responses may have limited activity against quiescent target cells. Therefore in order to cure the disease, specific activation strategies and/or prolonged persistence of specific T cells will be needed to achieve a potent anti-leukemic effect with controlled GVHD.

scholarly journals Blocking effect of lyt-2 antibodies on T cell functions.

1980 ◽  
Vol 152 (3) ◽  
pp. 674-687 ◽  
Author(s):  
N Hollander ◽  
E Pillemer ◽  
I L Weissman
Keyword(s):  
T Cells ◽  
T Cell ◽  
Mixed Lymphocyte Reaction ◽  
Cell Function ◽  
Mixed Lymphocyte Culture ◽  
Lymphocyte Culture ◽  
Target Cells ◽  
Cell Functions ◽  
T Cell Function ◽  
Helper Function

Monoclonal anti-Lyt-2 antibodies blocked effector function of cytotoxic thymus-derived (T) cells in the absence of added complement. Cytolysis of both allogeneic cells and syngeneic lymphoma or sarcoma target cells was inhibited at the level of the effector lymphocytes. Anti-Lyt-1 and anti-Thy-1 antibodies did not block killer cells. Proliferation of T cells in mixed lymphocyte culture was also inhibited by anti-Lyt-2, but not affected by anti-Lyt-1 or anti-Thy-1 antibodies. Although Lyt-1+ lymphocytes were required in the mixed lymphocyte reaction as helper cells for proliferation of Lyt-2+ lymphocytes, their helper function was not affected by the presence of Lyt-1 antibodies. Thus, although anti-Lyt-1, anti-Lyt-2 and anti-Thy-1 were of the same gamma 2A immunoglobulin class, had high titers, and interacted with T cells to the same extent, only anti-Lyt-2 blocked T cell functions. Polyclonal activation of T lymphocytes by concanavalin A, in contrast to activation by alloantigens, was not inhibited by Lyt-2 antibodies, suggesting that Lyt-2 antibodies interfere with T cell function at the level of the T cell antigen-receptor. The role which Lyt-2 molecules may play in T cell function is discussed.

scholarly journals A Novel T Cell-Engaging Bispecific Antibody for Treating Mesothelin-Positive Solid Tumors

Biomolecules ◽  
2020 ◽  
Vol 10 (3) ◽  
pp. 399
Author(s):  
Aerin Yoon ◽  
Shinai Lee ◽  
Sua Lee ◽  
Sojung Lim ◽  
Yong-Yea Park ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Solid Tumors ◽  
T Cell Activation ◽  
Target Cell ◽  
Cell Activation ◽  
Cell Killing ◽  
Bispecific Antibodies ◽  
Target Cells

As mesothelin is overexpressed in various types of cancer, it is an attractive target for therapeutic antibodies. T-cell bispecific antibodies bind to target cells and engage T cells via binding to CD3, resulting in target cell killing by T-cell activation. However, the affinity of the CD3-binding arm may influence CD3-mediated plasma clearance or antibody trapping in T-cell-containing tissues. This may then affect the biodistribution of bispecific antibodies. In this study, we used scFab and knob-into-hole technologies to construct novel IgG-based 1 + 1 MG1122-A and 2 + 1 MG1122-B bispecific antibodies against mesothelin and CD3ε. MG1122-B was designed to be bivalent to mesothelin and monovalent to CD3ε, using a 2 + 1 head-to-tail format. Activities of the two antibodies were evaluated in mesothelin-positive tumor cells in vitro and xenograft models in vivo. Although both antibodies exhibited target cell killing efficacy and produced regression of xenograft tumors with CD8+ T-cell infiltration, the antitumor efficacy of MG1122-B was significantly higher. MG1122-B may improve tumor targeting because of its bivalency for tumor antigen. It may also reduce systemic toxicity by limiting the activation of circulating T cells. Thus, MG1122-B may be useful for treating mesothelin-positive solid tumors.

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