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)

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.

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 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 Direct demonstration of the clonogenic potential of every human peripheral blood T cell. Clonal analysis of HLA-DR expression and cytolytic activity.

1983 ◽  
Vol 157 (2) ◽  
pp. 743-754 ◽  
Author(s):  
A Moretta ◽  
G Pantaleo ◽  
L Moretta ◽  
J C Cerottini ◽  
M C Mingari
Keyword(s):  
T Cells ◽  
T Cell ◽  
Peripheral Blood ◽  
Human Peripheral Blood ◽  
Cell Populations ◽  
Target Cells ◽  
Flow Cytofluorometry ◽  
Hla Dr ◽  
Clonogenic Potential ◽  
Precursor Frequency

In an attempt to determine the clonogenic properties of human peripheral blood T cells, we have developed a limiting dilution microculture system using phytohemagglutinin (PHA) as T cell activator and supernatant from PHA-stimulated spleen cultures as a source of T cell growth factors. The frequencies of cells capable of extensive proliferation under these culture conditions were 0.52-0.73, 0.98-1.11, and less than 0.02 in peripheral blood mononuclear, E-rosette-positive, and E-rosette-negative cell populations, respectively. The clonogenic potential of virtually all T cells was confirmed in experiments using single cells isolated by micromanipulation. Clone size ranged between 5 and 30 X 10(4) cells on day 14 of culture. The same microculture system was used to determine the precursor frequency of all cytolytic T lymphocytes (CTL-P). As assessed by a lectin-dependent 51Cr release assay, the CTL-P frequency in purified T cell populations ranged between 0.30 and 0.34. In comparison, the precursor frequency of T cells capable of lysing K562 target cells was ranging between 0.14 and 0.16. Parallel analysis of individual clonal cultures for both lytic activities showed that 50% of the clones exhibiting lectin-dependent lysis were also active against K562 target cells. All of the proliferating clones expressed HLA-DR antigens, although to a varying degree as assessed by flow cytofluorometry. Given the high cloning efficiency of this culture system, it appears now possible to determine the precursor frequencies of the various classes of functional cells in T cell populations.

AAV-2 Capsid-Specific CD8+ T Cells Limit the Duration of Gene Therapy in Humans and Cross-React with AAV-8 Capsid.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 455-455 ◽  
Author(s):  
Federico Mingozzi ◽  
Marcela V. Maus ◽  
Denise E. Sabatino ◽  
Daniel J. Hui ◽  
John E.J. Rasko ◽  
...  
Keyword(s):  
Gene Therapy ◽  
T Cells ◽  
T Cell ◽  
Cell Population ◽  
Cd8 T Cells ◽  
Peripheral Blood ◽  
Transgene Expression ◽  
Cd8 T Cell ◽  
Target Cells

Abstract Efforts to establish an adeno-associated viral (AAV) vector-mediated gene therapy for the treatment of hemophilia B have been hindered by an immune response to the viral capsid antigen. Preclinical studies in small and large animal models of the disease showed long-term factor IX (F.IX) transgene expression and correction of the phenotype. However, in a recent phase I/II clinical trial in humans (Manno et al., Nat. Med. 2006), after hepatic gene transfer with an AAV-2 vector expressing human F.IX transgene, expression lasted for only a few weeks, declining to baseline concurrently with a peak in liver enzymes. We hypothesized that T cells directed towards AAV capsid antigens displayed by transduced hepatocytes were activated and these mediated destruction of the transduced hepatocytes, thereby causing loss of transgene expression and a transient transaminitis. Peripheral blood mononuclear cells isolated from AAV-infused subjects were stained with an AAV capsid-specific MHC class I pentamer either directly or after in vitro expansion. Two weeks after vector infusion 0.14% of circulating CD8+ T cells were capsid-specific on direct staining, and five weeks after infusion the capsid-specific population had expanded to 0.5% of the circulating CD8+ T cells, indicating proliferation of this T cell subset. By 20 weeks after vector infusion, the capsid-specific CD8+ T cell population had contracted to the level seen at 2 weeks. The expansion and contraction of this capsid-specific CD8+ T cell population paralleled the rise and fall of serum transaminases in the subject observed. Subsequent ex vivo studies of PBMC showed the presence of a readily expandable pool of capsid-specific CD8+ T cells up to 2.5 years post vector-infusion. Similarly, we were able to expand AAV-specific CD8+ T cells from peripheral blood of normal donors, suggesting the existence of a T cell memory pool. Expanded CD8+ T cells were functional as evidenced by specific lysis of HLA-matched target cells and by IFN-γsecretion in response to AAV epitopes. It has been argued that potentially harmful immune responses could be avoided by switching AAV serotypes, however, capsid protein sequences are highly conserved among different serotypes, as are some immunodominant epitopes that we identified. Indeed, we demonstrated that capsid-specific CD8+ T cells from AAV-infused hemophilic subjects functionally cross-react with AAV-8. Moreover, cells expanded from normal donors with AAV-2 vector capsids proliferated upon culture with AAV-8 capsids, demonstrating that both vectors could be processed appropriately in vitro to present the epitopic peptide to capsid-specific T cells. This suggests that AAV-2-specific memory CD8+ T cells normally present in humans likely would expand upon exposure to AAV-8 capsid epitopes. We conclude that the use of immunomodulatory therapy may be a better approach to achieving durable transgene expression in the setting of AAV-mediated gene therapy.

WT1-Specific T Cells Exhibiting a Non-Exhausted, Functional Phenotype Can Be Generated From The Natural Repertoire Of Healthy Donors For Clinical Use

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 4504-4504 ◽  
Author(s):  
Sabine Schmied ◽  
Anne Richter ◽  
Mario Assenmacher ◽  
Juergen Schmitz
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cd8 T Cells ◽  
Cd4 T Cells ◽  
Ex Vivo ◽  
Leukemic Cells ◽  
Target Cells ◽  
Clinical Use ◽  
Healthy Donors ◽  
Effector Cytokines

Background The Wilms tumor antigen 1 (WT1) is a self-antigen expressed at high levels in leukemic cells, but not in healthy tissue. As WT1 expression in leukemic cells drives leukemogenesis, it is a favorable target antigen for immunotherapy, e.g. adoptive transfer of allogeneic T cells, to prevent or treat leukemic relapse after stem cell transplantation (Cheever et al., Clin Cancer Res 2009;15(17)). WT1-specific CD8+ T cells have been detected in healthy individuals at low frequencies (Rezvani et al., Blood 2003;102). However, a comprehensive characterization of CD4+ and CD8+WT1-specific T cells is missing and the efficient expansion of a polyclonal WT1-reactive T cell population for clinical use has remained a major challenge. In this study we aim to directly ex vivo characterize WT1-specific T cells present in the blood of healthy donors at high-resolution and to develop a rapid method for the generation of functionally potent, polyclonal CD4+ and CD8+WT1-specific T cells for clinical use. Methods For direct ex vivo analysis of CD4+ WT1-specific T cells peripheral blood mononuclear cells (PBMC) of healthy blood donors were in vitro stimulated with a pool of overlapping peptides spanning the WT1 protein for 7 hours. Subsequently CD154 (CD40L)-expressing cells were magnetically enriched and flow cytometrically examined for expression of effector cytokines and their differentiation status. Presence and phenotype of CD8+ WT1-specific T cells have been studied after stimulation of presorted naïve and memory T cell populations with WT-1 peptide pool for 30 hours, magnetic enrichment of CD137+ (4-1BB) cells and subsequent staining using pMHCI-Tetramers. For the generation of polyclonal WT1-specific CD4+ and CD8+ T cells PBMC were in vitro activated with WT-1 peptide pool for 30 hours. CD137+cells were magnetically selected and expanded for 9 days in the presence of the cytokines IL-7, IL-15 and IL-21 at low doses. Expanded T cells were analyzed for their phenotype, the expression of co-stimulatory and exhaustion markers and were tested for their functionality and cytotoxicity by restimulation experiments with antigen-loaded target cells. Results Ex vivo frequencies of WT1-specific T cells are low, 1 to 10 WT1-specific CD154+ CD4+ T cells can be detected within 1x106 CD4+ T cells. In about 80% of healthy donors (n=15) a CD4+ memory response, accompanied by production of effector cytokines like IFNγ, TNFα and IL-2, against WT1 peptides is present. Additionally, in all donors naïve WT1-specific CD4+ T cells can be detected. In contrast, detected CD137+CD8+ WT1-reactive T cells exhibit a naïve phenotype (CD45RA+CCR7+) in all donors (n=5), no WT1-reactive CD8+T cells could be enriched from presorted memory T cells. To evaluate the usefulness of our improved short-term expansion protocol to generate potent WT1-specific T cell cultures for clinical use, we characterized CD137 enriched and expanded T cells. Notably, a high frequency of CD4+ and CD8+ T cells show specific reactivity against WT1-presenting autologous cells as detected by production of effector cytokines like IFNγ, TNFα and IL-2 after antigen-specific restimulation. Cytotoxic activity against antigen-loaded target cells could be shown by direct flow-cytometry-based cytotoxicity assays and antigen-specific upregulation of the degranulation marker CD107a. Stainings using multiple WT1-MHCI-tetramers furthermore confirmed antigen-specificity and suggested polyclonality within the CD8+T cell population. In contrast to previous expansion protocols our polyclonally expanded T cells exhibit a favourable, unexhausted memory phenotype, express co-stimulatory markers CD27 and CD28 and the IL7R-a chain (CD127) which has been shown to mark cells with stem T cell like properties. Furthermore exhaustion markers like CD279 (PD-1), CD178 (FasL) and CD57 are scarcely expressed. Conclusions Functional, polyclonal, CD4+ and CD8+ WT1-specific, reactive T cells can be efficiently enriched directly ex vivo from the natural repertoire by magnetic separation of T cells after antigen-specific stimulation. Phenotypic and functional characterization revealed a non-exhausted phenotype of expanded WT1-specific T cells, thereby suggesting good persistence and functionality of the obtained T cell product in vivo. Thus, our approach holds great potential for the GMP-compliant generation of WT1-specific T cells for future clinical use. Disclosures: Schmied: Miltenyi Biotec GmbH: Employment. Richter:Miltenyi Biotec GmbH: Employment. Assenmacher:Miltenyi Biotec GmbH: Employment. Schmitz:Miltenyi Biotec: Employment.

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.

scholarly journals Targeting BTN2A1 By a Unique Activating Mab Improves Vγ9Vδ2 T Cell Cytotoxicity Against Primary Acute Lymphoblastic Blasts

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 2302-2302
Author(s):  
Anne-Charlotte Le Floch ◽  
Caroline Imbert ◽  
Aude De Gassart ◽  
Florence Orlanducci ◽  
Aude Le Roy ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Board Of Directors ◽  
Cell Lines ◽  
Research Funding ◽  
Leukemic Cells ◽  
Target Cells ◽  
Advisory Committees ◽  
Vγ9vδ2 T Cells ◽  
Privately Held

Abstract Introduction Vγ9Vδ2 T cells are new promising cytotoxic effectors in hematological malignancies. In acute myeloid leukemia and in non-Hodgkin lymphomas, Vγ9Vδ2 T cells-based immunotherapy has shown encouraging results both in preclinical models and in early phase clinical trials. Acute lymphoblastic leukemia (ALL) includes very heterogeneous clinico-biological entities, for which recent immunotherapy approaches are currently being developed. Nevertheless, global prognosis of ALL patients still be poor with a 5 years-overall survival of less than 40% and therefore, treatments need to be improved. Very few data are currently available on susceptibility of ALL blasts to Vγ9Vδ2 T cell cytotoxic activity. Vγ9Vδ2 T cells are activated by phosphoantigens bound to BTN3A1 on target cells. BTN3A molecules are targeted at clinical level, with the ICT01 agonist monoclonal antibody (mAb), that is currently tested in a multicentric phase ½ study (EVICTION study). Biology of Vγ9Vδ2 T cells has recently undergone a new paradigm with the identification of BTN2A1 as the direct ligand for Vγ9 chain of γδ TCR. BTN2A1 is mandatory for Vγ9Vδ2 T cell activation but its precise role in modulating functions of Vγ9Vδ2 T cells remains unknown. Here, we show that allogenic and autologous Vγ9Vδ2 T cells exert cytolytic functions against ALL cell lines and primary ALL blasts, and we report that Vγ9Vδ2 T cell cytotoxic activity is enhanced after treatment with a unique agonist mAb targeting BTN2A1. Material and methods 5 ALL cell lines (697, RS4;11, NALM-6, HPB-ALL, SUP-T1) and PBMC from 11 adults ALL patients at diagnosis (B-ALL, T-ALL and Ph+ ALL) were tested in functional assays. We evaluated apoptosis of ALL cell lines and of primary ALL blasts after coculture with allogenic Vγ9Vδ2 T cells. ALL samples were also tested for their expansion capacities and a degranulation assay was performed at D14. We assessed in parallel relative quantification of the level expression of BTN2A1 (ICT0302 and 7.48 epitopes), and BTN3A (20.1 and 108.5 epitopes) on surface of ALL blasts. DAUDI-BTN2AKO+2A1 and HEK293-BTN2AKO+2A1 cells were used in binding assays, and modulation of TCR binding was assessed using recombinant tetramerized Vγ9Vδ2 TCR. Results We showed that Vγ9Vδ2 T cells exert spontaneous cytotoxicity against ALL cell lines and primary ALL blasts with a heterogeneous susceptibility depending on the target. We demonstrated that anti-BTN2A1 ICT0302 agonist mAb significantly enhanced Vγ9Vδ2 T cells mediated apoptosis in comparison to control condition, even for the less spontaneously susceptible cells. We confirmed these observations with degranulation of autologous Vγ9Vδ2 T cells expanded from 5 ALL patients at diagnosis that was increased after treatment with anti-BTN2A1 ICT0302 agonist mAb. BTN3A and BTN2A1 were detected on surface of ALL blasts, and BTN3A 108.5 was the most expressed epitope. Interestingly, we observed that anti-BTN2A1 ICT0302 strongly increased binding of a recombinant Vγ9Vδ2 TCR to target cells using with HEK293 and DAUDI cells. Discussion Our results highlighted that Vγ9Vδ2 T cells exert cytolytic functions against ALL cells, both in allogenic and autologous setting and demonstrated that BTN2A1 targeting with our unique agonist mAb could potentiate effector activities of Vγ9Vδ2 T cells against ALL blasts. These results indicate that the sensitization of leukemic cells can be induced by activation BTN3A as well as BTN2A1 mAbs. These data bring novel understanding on the biology of BTN2A1 on leukemic cells and our ability to enhance both binding and function. These findings could be of great interest for the design of innovative Vγ9Vδ2 T cells-based immunotherapy strategies for treating ALL that could be extended to other cancer types. Disclosures De Gassart: ImCheck Therapeutics: Current Employment, Current holder of individual stocks in a privately-held company. Vey: Amgen: Honoraria; BMS: Honoraria; BIOKINESIS: Consultancy, Research Funding; NOVARTIS: Consultancy, Honoraria, Research Funding; SERVIER: Consultancy; JAZZ PHARMACEUTICALS: Honoraria; JANSSEN: Consultancy. Cano: ImCheck Therapeutics: Current Employment, Current holder of individual stocks in a privately-held company. Olive: Emergence Therapeutics: Current holder of individual stocks in a privately-held company, Membership on an entity's Board of Directors or advisory committees; Alderaan Biotechnology: Current holder of individual stocks in a privately-held company, Membership on an entity's Board of Directors or advisory committees; ImCheck Therapeutics: Current holder of individual stocks in a privately-held company, Membership on an entity's Board of Directors or advisory committees. OffLabel Disclosure: Anti-BTN2A1 ICT0302 is a murine agonist monoclonal antibody targeting BTN2A1 whose aim is to increase Vgamma9Vdelta2 T cells functions.

Generation of GMP-Grade CMV pp65-Specific CD8+ and CD4+ Donor T Cell Lines for Treatment of CMV Reactivation after Transplantation.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 2931-2931
Author(s):  
Maarten L. Zandvliet ◽  
J.H. Frederik Falkenburg ◽  
Inge Jedema ◽  
Roelof Willemze ◽  
Henk-Jan Guchelaar ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Lines ◽  
Cd4 T Cells ◽  
Target Cells ◽  
Specific Cell ◽  
Specific Response ◽  
Specific Lysis ◽  
T Cell Lines

Abstract Reactivation of CMV remains a major cause of morbidity and mortality in immunocompromised recipients of allogeneic stem cell transplantation. Antiviral pharmacotherapy may not be sufficient due to significant toxicity and moderate efficacy. It has been shown that adoptive transfer of donor-derived CMV-specific T cells may be an effective strategy to control established CMV infection. For a persistent function in vivo the presence of both virus-specific CD8+ and CD4+ T cells is essential. Therefore, we developed an optimized protocol for the generation of CMV pp65-specific CD8+ and CD4+ T cell lines which is fully compliable with Good Manufacturing Practice (GMP) conditions. Enrichment for CMV-specific T cells followed by only a short culture period is likely to retain maximal in vivo potential. PBMCs from 7 CMV seropositive donors were stimulated with recombinant pp65 protein (7–70 μg/ml) and/or HLA-A*0201/HLA-B*0702 restricted immunodominant pp65 peptides (NLV/TPR). Peptides used were clinical grade, and recombinant protein was gamma-irradiated (50 kGy, −80 C°) to eliminate possible microbiological contamination. High dose gamma-irradiation of pp65 protein resulted in partial degradation, but antigenic presentation was maintained. IFNγ producing cells were enriched using the IFNγ secretion assay (Miltenyi Biotec) at day 1 after stimulation, and cultured with autologous feeders (10x) and IL-2 (10 or 50 IU IL-2/ml) with or without CD3/28 expansion beads. Addition of high concentrations of protein during initial stimulation had a negative effect on enrichment probably due to non-specific stimulation of cells. Addition of immunodominant pp65 peptides promoted isolation efficiency and proliferation of epitope-specific CD8+ T cells in some donors. Cell lines were analyzed at different time points (day 4–15) using peptide-MHC tetramer and phenotypic markers. In addition, pp65-specificity was evaluated by intracellular IFNγ staining after restimulation with a pp65 protein-spanning pool of 15-mer peptides. CMV-specific lysis was tested in a 51-Cr release assay on pp65-transduced target cells. Enrichment of IFNγ producing cells after pp65 protein stimulation resulted in pp65-specific cell lines consisting of both CD8+ and CD4+ T cells. The T cell subset distribution directly after enrichment did not change during culture and was reproducible for each donor. Moreover, the composition of T cell lines reflected the pp65-specific response in donor PBMC starting material. The CD8+ compartment contained the known immunodominant tetramer staining cells (range 5–100%). The majority of both CD8+ and CD4+ T cells produced IFNγ upon restimulation with the pp65 peptide-pool, and showed CMV-specific lysis of target cells. The phenotype of pp65-specific T cells was predominant CD28+/CD45RO+ and CD45RA−/CCR7−/CD62L−, although CCR7 and CD62L were transiently expressed at day 4 and 7 after stimulation. Cryopreservation did not affect the composition or functionality of T cell lines. In conclusion, this procedure yields GMP-grade T cell lines comprising both CD8+ and CD4+ CMV-specific T cells. Processing and presentation of CMV protein by donor antigen-presenting cells enables selection of the full pp65-specific donor repertoire, without restrictions related to HLA or known epitopes. The choice for a moderate or more vigorous expansion after enrichment remains arbitrary and needs to be evaluated in clinical trials.

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.

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