scholarly journals A negative role for the interleukin-2-inducible T-cell kinase (ITK) in human Foxp3+ TREG differentiation

2018 ◽  
Author(s):  
Polina Mamontov ◽  
Ryan A. Eberwine ◽  
Jackie Perrigoue ◽  
Anuk Das ◽  
Joshua R. Friedman ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
T Cell Activation ◽  
Cell Activation ◽  
Kinase Inhibitor ◽  
Interleukin 2 ◽  
Tec Kinases ◽  
Human T Cells ◽  
Inducible T Cell Kinase

ABSTRACTThe Tec kinases ITK (interleukin-2-inducible T-cell kinase) and RLK (resting lymphocyte kinase) are critical components of the proximal TCR/CD3 signal transduction machinery, and data in mice suggest that ITK negatively regulates TREG differentiation. However, whether Tec kinases modulate TREG development and/or function in human T cells remains unknown. Using a novel self-delivery siRNA platform (sdRNA), we found that ITK knockdown in primary human naïve peripheral blood CD4 T cells increased Foxp3+ TREG differentiation under both TREG and T effector (Teff) cell priming conditions. ITK knockdown also enhanced the expression of the co-inhibitory receptor PD-1 on FoxP3+ T cells. TREGS differentiated in vitro (iTREG) after ITK knockdown displayed suppressive capacity against effector CD4+ T cell proliferation. ITK knockdown decreased IL-17A production in T cells primed under Th17 conditions and increased Th1 differentiation. Finally, a dual ITK/RLK Tec kinase inhibitor blocked TREG differentiation and T cell activation in general. Our data suggest that targeting ITK in human T cells may be an effective approach to boost TREG in the context of autoimmune diseases, but non-specific inhibition of other Tec family kinases may broadly inhibit T cell activation.

scholarly journals Interleukin-2-dependent phosphorylation of the retinoblastoma-susceptibility-gene product p110-115RB in human T-cells

1992 ◽  
Vol 282 (3) ◽  
pp. 759-764 ◽  
Author(s):  
G A Evans ◽  
L M Wahl ◽  
W L Farrar
Keyword(s):  
Cell Cycle ◽  
T Cells ◽  
T Cell ◽  
Gene Transcription ◽  
T Cell Activation ◽  
Cell Activation ◽  
Susceptibility Gene ◽  
Interleukin 2 ◽  
Human T Cells ◽  
Rb Phosphorylation

The state of phosphorylation of the retinoblastoma-susceptibility gene product, p110-115RB, is thought to have fundamental importance in controlling the progression of the cell through the cell cycle. We have studied RB phosphorylation in human T-cells in the context of T-cell activation, stimulated by phytohaemagglutinin (PHA) and interleukin-2 (IL-2). We show that, of the signals associated with T-cell activation, only signals that directly lead to movement into S phase of the cell cycle are capable of stimulating RB phosphorylation. Cyclosporin A (CsA), a potent inhibitor of IL-2 synthesis and cellular proliferation, blocked RB phosphorylation, and this was recovered with exogenous IL-2, indicating a direct involvement of IL-2 in controlling RB phosphorylation. We found that PHA did not stimulate RB phosphorylation within 10 h of treatment, but IL-2 could effectively stimulate RB phosphorylation within 2 h, and this approached a maximum within 8-10 h of IL-2 treatment. Further, by using actinomycin D to inhibit new gene transcription following IL-2 stimulation, we found that early-cell-cycle phosphorylation of RB required IL-2-stimulated gene transcription. From these data we conclude that, in human T-cells, RB phosphorylation is not directly associated with T-cell receptor-mediated events, but requires the interaction of IL-2 and new gene transcription following IL-2 stimulation.

scholarly journals The immunobiology of CD27 and OX40 and their potential as targets for cancer immunotherapy

Blood ◽  
2018 ◽  
Vol 131 (1) ◽  
pp. 39-48 ◽  
Author(s):  
Sarah L. Buchan ◽  
Anne Rogel ◽  
Aymen Al-Shamkhani
Keyword(s):  
T Cells ◽  
T Cell ◽  
T Cell Activation ◽  
Cell Activation ◽  
High Profile ◽  
Cell Immunity ◽  
Human T Cells ◽  
Checkpoint Receptors ◽  
Tumor Types

In recent years, monoclonal antibodies (mAbs) able to reinvigorate antitumor T-cell immunity have heralded a paradigm shift in cancer treatment. The most high profile of these mAbs block the inhibitory checkpoint receptors PD-1 and CTLA-4 and have improved life expectancy for patients across a range of tumor types. However, it is becoming increasingly clear that failure of some patients to respond to checkpoint inhibition is attributable to inadequate T-cell priming. For full T-cell activation, 2 signals must be received, and ligands providing the second of these signals, termed costimulation, are often lacking in tumors. Members of the TNF receptor superfamily (TNFRSF) are key costimulators of T cells during infection, and there has been an increasing interest in harnessing these receptors to augment tumor immunity. We here review the immunobiology of 2 particularly promising TNFRSF target receptors, CD27 and OX40, and their respective ligands, CD70 and OX40L, focusing on their role within a tumor setting. We describe the influence of CD27 and OX40 on human T cells based on in vitro studies and on the phenotypes of several recently described individuals exhibiting natural deficiencies in CD27/CD70 and OX40. Finally, we review key literature describing progress in elucidating the efficacy and mode of action of OX40- and CD27-targeting mAbs in preclinical models and provide an overview of current clinical trials targeting these promising receptor/ligand pairings in cancer.

scholarly journals Delivery of IL-2 to the T Cell Surface Through Phosphatidylserine Permits Robust Expansion of CD8 T Cells

2021 ◽  
Vol 12 ◽  
Author(s):  
Alana MacDonald ◽  
Brandon Lam ◽  
John Lin ◽  
Louise Ferrall ◽  
Yu Jui Kung ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Surface ◽  
T Cell Activation ◽  
Cd8 T Cells ◽  
Cell Activation ◽  
Interleukin 2 ◽  
Annexin V ◽  
Cell Immunity

The phospholipid phosphatidylserine (PS) is naturally maintained on the cytoplasmic side of the plasma membrane. Independent of apoptosis, PS is redistributed to the surface of CD8 T cells in response to TCR-mediated activation. Annexin V (AnnV) is a protein known to bind PS with high affinity and has been effectively utilized to anchor antigen to the surface of CD8 T cells. To expand these studies, we aimed to exploit TCR activation driven PS exposure as a target to deliver cytokine, namely interleukin-2 (IL-2), to the surface of CD8 T cells. This was accomplished using a novel chimeric fusion protein of annexin V and interleukin 2 (AnnV-IL2). In vitro analysis revealed that AnnV-IL2 is able to specifically bind PS on the T cell surface following TCR stimulation. Consequently, AnnV-IL2 proved to be significantly more effective at enhancing T cell activation compared to recombinant IL-2. In vivo, AnnV-IL2 promotes robust expansion of antigen-specific cells capable of interferon gamma (IFNγ) production when administered following peptide vaccination. Importantly, upon antigen rechallenge, AnnV-IL2 treatment mice demonstrated a stronger secondary expansion, indicating durability of AnnV-IL2 mediated responses. Our data supports the use of AnnV-IL2 to modulate antigen-specific T cell immunity and demonstrates that the PS-AnnV axis is a feasible mechanism to target diverse cargo to CD8 T cells.

scholarly journals Inhibition of Vasoactive Intestinal Peptide Signaling with More Potent Inhibitors Augments T-Cell Activation and Prolongs Survival in Leukemic Mice

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 1868-1868
Author(s):  
Tenzin Passang Fnu ◽  
Jianming Li ◽  
Sruthi Ravindranathan ◽  
Edmund K. Waller
Keyword(s):  
T Cells ◽  
Amino Acid ◽  
T Cell ◽  
T Cell Activation ◽  
Cell Activation ◽  
Second Generation ◽  
The Novel ◽  
Human T Cells

Abstract Introduction: Vasoactive intestinal peptide (VIP) is a 28-amino acid neuropeptide with immunosuppressive effects on T cells. Inhibition of VIP receptor (VIP-R) signaling by VIPhyb, a first-generation VIP-R antagonist, not only enhances T-cell activation and proliferation in vitro but also improves T cell dependent anti-tumor response in mouse models of acute myeloid leukemia (AML) and T lymphoblastic leukemia (Li et al. 2016; Petersen, Li, and Waller 2017). The goal of the project is to develop more potent VIP-R antagonists that generate a significantly more robust anti-tumor response in mouse models of AML, when compared to VIPhyb and validate a screening method to test the efficacy of novel peptides in activating human T cells in vitro. In this study, we report, for the first time, the activity of novel VIP-R antagonists on the activation profile of human T cells. Methods: We utilized in-silico-based modeling to identify 10 novel VIP-R antagonists from a library of 300 peptide sequences predicted to have increased binding affinity to VIP receptors VPAC1 and VPAC2 when compared to VIP or VIPhyb (Table 1). The library was generated from peptide sequences that contain the six charged N-terminal residues of the neurotensin present in VIPhyb with two or more amino acid substitutions within the C-terminal amino acid sequence of VIP. The ability of these peptides was tested in vitro using T cells from multiple healthy human donors activated using anti-CD3 monoclonal antibody coated plates. Activation status was assessed by flow cytometry of CD69, OX40, PD1, Tim3 and Lag3 expression relative to control cultures without added peptides. Potency of the novel antagonists in vivo was tested in a mouse AML model, by treating C1498- bearing mice with subcutaneous administration of VIP, VIPhyb, scrambled peptide (SCRAM1) or the second-generation VIP-R antagonists (labeled as 'ANT') from day 6-12 after tumor implantation. Results: Inhibiting VIP-R signaling in human T cells using second-generation VIP-R antagonists ANT008, ANT308 and ANT195 showed approximately 1.5-to-2-fold increase in CD69, OX40, Tim3, Lag3 and OX40 expression in CD4+ T cells following 24-hour of drug exposure compared to control cultures (Figure 1A). A smaller effect of VIP-R antagonists on activation of CD8+ subsets was observed (Figure 1B). Among the peptides, ANT195 was superior to ANT008 and ANT308 which shows potency even at 1μM compared to 3μM for ANT008 and ANT308. However, significant increase in CD69 expression was observed in both CD4+ and CD8+ T cells in cultures treated with ANT308 (Figure 1 A&B, *p<0.05). Viability of the T cells was not affected by incubation with the queried peptides (Data not shown). These data corresponded to in vivo activity of the novel VIP-R antagonists such as ANT308 and ANT195 which rendered 40% of mice leukemia-free at day 60 compared to only 5% long-term survival with VIPhyb (Figure 2). Another candidate, ANT300, increased median survival time (MST) by up to 47 days compared to MST of 34 days with VIPhyb (Figure 2). Conclusions: Here, we report a simple and robust in vitro method to screen for immune activity potential of novel second-generation VIP-R antagonists using human T cells. Preliminary screen shows VIP-R antagonists augment activation of both CD4+ and CD8+ T cells. Our results indicate that ANT308 and ANT195 are more potent VIP-R antagonists with enhanced activity in vitro (human) and in vivo (mouse) than VIPhyb and ANT008, which demonstrate lower predicted binding affinities to VPAC1 and VPAC2. Our study supports the hypothesis that higher predicted binding affinity to VPAC1 and/or VPAC2 is associated with enhanced activity in stimulating human T cells and promoting anti-leukemia activity in mice. Further mechanistic studies on how inhibition of VIP-R signaling augments T cell activation and function are underway. These novel antagonists can lead to peptide-based immunotherapy for the treatment of various liquid cancers. Clinical development of this novel concept will require appropriate pre-clinical pharmacokinetic and toxicology studies. Figure 1 Figure 1. Disclosures Waller: Cambium Oncology: Current holder of individual stocks in a privately-held company, Current holder of stock options in a privately-held company; Verastem Oncology: Consultancy, Research Funding.

scholarly journals Differential Interleukin-2 Transcription Kinetics Render Mouse but Not Human T Cells Vulnerable to Splicing Inhibition Early after Activation

2019 ◽  
Vol 39 (16) ◽  
Author(s):  
Debojit Bose ◽  
Alexander Neumann ◽  
Bernd Timmermann ◽  
Stefan Meinke ◽  
Florian Heyd
Keyword(s):  
T Cells ◽  
T Cell ◽  
T Cell Activation ◽  
Cell Activation ◽  
De Novo ◽  
Time Window ◽  
Interleukin 2 ◽  
Adaptive Immune Response ◽  
Human T Cells ◽  
Splicing Efficiency

ABSTRACTT cells are nodal players in the adaptive immune response against pathogens and malignant cells. Alternative splicing plays a crucial role in T cell activation, which is analyzed mainly at later time points upon stimulation. Here we have discovered a 2-h time window early after stimulation where optimal splicing efficiency or, more generally, gene expression efficiency is crucial for successful T cell activation. Reducing the splicing efficiency at 4 to 6 h poststimulation significantly impaired murine T cell activation, which was dependent on the expression dynamics of the Egr1–Nab2–interleukin-2 (IL-2) pathway. This time window overlaps the time of peak IL-2de novotranscription, which, we suggest, represents a permissive time window in which decreased splicing (or transcription) efficiency reduces mature IL-2 production, thereby hampering murine T cell activation. Notably, the distinct expression kinetics of the Egr1–Nab2–IL-2 pathway between mouse and human render human T cells refractory to this vulnerability. We propose that the rational temporal modulation of splicing or transcription during peakde novoexpression of key effectors can be used to fine-tune stimulation-dependent biological outcomes. Our data also show that critical consideration is required when extrapolating mouse data to the human system in basic and translational research.

scholarly journals Autologous Tumor Infiltrating T Cells Cytotoxic for Follicular Lymphoma Cells Can Be Expanded In Vitro

Blood ◽  
1997 ◽  
Vol 89 (10) ◽  
pp. 3806-3816 ◽  
Author(s):  
Joachim L. Schultze ◽  
Mark J. Seamon ◽  
Sabine Michalak ◽  
John G. Gribben ◽  
Lee M. Nadler
Keyword(s):  
T Cells ◽  
T Cell ◽  
T Cell Activation ◽  
Cell Activation ◽  
Cytotoxic T Cells ◽  
Interleukin 2 ◽  
Interferon Γ ◽  
Autologous Tumor

Abstract Follicular lymphomas (FLs) rarely induce clinically significant T-cell–mediated responses. We showed that freshly isolated tumor infiltrating T cells (T-TILs) lack tumor-specific cytotoxicity. Stimulation of these T cells with FL cells in the presence of interleukin-2 (IL-2) and/or costimulation via CD28 does not lead to T-cell activation and expansion. In contrast, when stimulated with FL cells preactivated via CD40, autologous T-TILs can be expanded by the addition of exogenous IL-2. These T cells can be further expanded in vitro by the addition of exogenous IL-4, IL-7, or interferon-γ, but not IL-12. Once activated, these T cells showed FL-directed cytotoxicity in four of five patients tested. We concluded that autologous cytotoxic anti-FL–specific T cells exist, but can only be detected in vitro under optimized conditions for T-cell stimulation and expansion. This suggests that their frequency in vivo is either very low or that the microenvironment does not provide the necessary signals to activate these T cells. This model system allows dissection of the requisite conditions for activation and expansion of lymphoma-directed cytotoxicity and may permit expansion of previously activated cytotoxic T cells for adoptive transfer.

scholarly journals CD4+CD25+ Immunoregulatory T Cells Suppress Polyclonal T Cell Activation In Vitro by Inhibiting Interleukin 2 Production

1998 ◽  
Vol 188 (2) ◽  
pp. 287-296 ◽  
Author(s):  
Angela M. Thornton ◽  
Ethan M. Shevach
Keyword(s):  
T Cells ◽  
T Cell ◽  
T Cell Activation ◽  
Cell Activation ◽  
Interleukin 2 ◽  
Peripheral Tolerance ◽  
Cell Contact ◽  
Suppressor T Cells

Peripheral tolerance may be maintained by a population of regulatory/suppressor T cells that prevent the activation of autoreactive T cells recognizing tissue-specific antigens. We have previously shown that CD4+CD25+ T cells represent a unique population of suppressor T cells that can prevent both the initiation of organ-specific autoimmune disease after day 3 thymectomy and the effector function of cloned autoantigen-specific CD4+ T cells. To analyze the mechanism of action of these cells, we established an in vitro model system that mimics the function of these cells in vivo. Purified CD4+CD25+ cells failed to proliferate after stimulation with interleukin (IL)-2 alone or stimulation through the T cell receptor (TCR). When cocultured with CD4+CD25− cells, the CD4+CD25+ cells markedly suppressed proliferation by specifically inhibiting the production of IL-2. The inhibition was not cytokine mediated, was dependent on cell contact between the regulatory cells and the responders, and required activation of the suppressors via the TCR. Inhibition could be overcome by the addition to the cultures of IL-2 or anti-CD28, suggesting that the CD4+CD25+ cells may function by blocking the delivery of a costimulatory signal. Induction of CD25 expression on CD25− T cells in vitro or in vivo did not result in the generation of suppressor activity. Collectively, these data support the concept that the CD4+CD25+ T cells in normal mice may represent a distinct lineage of “professional” suppressor cells.

Targeting CD123 In Leukemic Stem Cells Using Dual Affinity Re-Targeting Molecules (DARTs®)

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 360-360 ◽  
Author(s):  
Muneera H. AL Hussaini ◽  
Julie Ritchey ◽  
Michael P. Rettig ◽  
Linda Eissenberg ◽  
Geoffrey L. Uy ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Tumor Cells ◽  
T Cell Activation ◽  
Peripheral Blood ◽  
Cell Activation ◽  
Extracellular Domain ◽  
Nsg Mice ◽  
Human T Cells

Abstract T-cell directed killing of tumor cells using bispecific reagents is a promising approach for the treatment of hematologic malignancies. In contrast to B-cell malignancies, this approach has been limited in AML by the lack of tumor-specific antigens/targets. CD123 (IL3RA) is highly and differentially expressed in AML blasts compared to normal hematopoietic stem and progenitor cells and is a potential target for immunotherapy. We investigated the ability of a DART constructed from an antibody to CD123 (7G3) and a MacroGenics’ proprietary CD3 antibody to redirect T cells against CD123+ AML blasts. DARTs consist of 2 independent polypeptides, each comprising the VH of one antibody in tandem with the VL of the other antibody. The resultant heterodimer is stabilized by a disulfide bond at the carboxyl terminal domains of the 2 VH regions. This construct binds to both the N-terminal extracellular domain of human CD123 and to the extracellular domain of CD3 in the human T-cell receptor complex. Our in vitro studies demonstrate that the CD3xCD123 DART induces specific aggregation of TCR/CD3+ Jurkat or human T cells and human CD123-transduced K562 (K562CD123/GFP) cells compared to a control DART lacking specificity for one of the antigens (16±3.2% vs. 1.6±0.2%, p=0.0074) or when compared to CD3xCD123 DART incubated with control GFP-transduced K562 (K562GFP) cells. Incubation of human T cells (1:1 ratio) with K562CD123/GFP and CD3xCD123 DART vs. control DARTS (10 ng/ml) for 5 days in vitro resulted in profound T-cell activation (CD25 expression, 88.8±2.7% vs.1.2±0.2%; p=0.0009), T-cell proliferation (VPD-450 proliferation assay; 98.2 ± 0.4% vs. 2.27± 0.4%, p=0.0001), expansion of the central memory T cell compartment (TCM, 62.6±1.5% vs. 5±1.3%, p<0.0001), and killing of K562CD123/GFP targets as measured by 7-AAD FACS (97±0.9% apoptosis relative to the control; p<0.0001) and chromium release assays (28.5% vs.3.1%; p=0.0002). As expected, aggregation, T-cell activation and target cell killing was negligible when T cells and CD3xCD123 DART were incubated with control K562GFP cells. Similar results were seen when A20 targets (BALB-C/H-2d B lymphoma cell line) overexpressing CD123 were used (7-AAD+ after 18 hours, 91.1±2.01% vs. 28.1±0.76%, p=0.0012). In spite of very low E:T ratios (0.009:1-0.071:1), when primary frozen/thawed AML peripheral blood specimens (n=6; CD123+ blasts ranging from 34.7 to 87.2%) were used, there was a profound and universal CD3xCD123 DART-specific activation and expansion of the few human T cells present in these AML samples (ranging between 0.9 to 6.6% of cells). After 6 days of in vitro incubation (37°C w/o exogenous IL-2) of each AML sample (1x106/ml) with CD3xCD123 DART (0.1 ng/ml) or control DARTs, there was a CD3xCD123 DART-specific increase in both T cell numbers (median: 8 fold, range:1.7-15 fold vs. 0.6-1 fold for control DARTs), and in the percentage of T cells expressing CD25 (median: 69.4 fold, range: 21.4-152.8 fold vs. 0.2-6.5 fold for control DARTs). This was accompanied by a dose-dependent reduction in blasts by 35±25% at 0.1 ng/ml DART and 99.6±0.05% at 10 ng/ml DART (p=0.0063). In addition, AML colony-forming units (L-CFU) were also inhibited by 94 ±0.6% while CFU-GEMM, CFU-GM and BFU-E from cord blood and (G-CSF)-mobilized peripheral blood from normal donors were not affected by either CD3xCD123 DART or control DARTs (0.1-10 ng/ml), suggesting limited impact on normal human hematopoietic progenitors in vitro. Bioluminescence imaging of irradiated NSG mice (n= 5/group; 300cGy) performed on days 3, 12, 19, and 28 after the infusion of 1.5 x 106 Click Beetle Red (CBR) luciferase+- transduced K562CD123/GFP cells on day 0 and both CD3xCD123 DART (0.5 mg/kg IV) and human T cells (3 x 106) on day 3 revealed no expansion of tumor cells in sharp contrast to NSG mice receiving either control DARTs and/or no human T cells (1415- fold expansion; p<0.0001). Of interest is that at 6 weeks post-infusion of primary AML xenografts into NSG mice (E:T=0.009:1), there was near- complete elimination (>97%) of AML blasts from the peripheral blood even in the absence of exogenously added human T cells. Clearing of primary human AML blasts from the spleen and bone marrow was also significant (40-77.8%) but less than that seen in the blood. These results provide the basis for the CD3xCD123 DART as a novel reagent for the treatment of patients with CD123+ AML. Disclosures: Chichili: Macrogenics. Inc: Employment. Moore:Macrogenics. Inc.: Employment. Johnson:Macrogenics. Inc.: Employment. Bonvini:Macrogenics. Inc.: Employment.

Quiescent phenotype of tumor-specific CD8+ T cells following immunization

Blood ◽  
2004 ◽  
Vol 104 (7) ◽  
pp. 1970-1978 ◽  
Author(s):  
Vladia Monsurrò ◽  
Ena Wang ◽  
Yoshisha Yamano ◽  
Stephen A. Migueles ◽  
Monica C. Panelli ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
T Cell Activation ◽  
Cd8 T Cells ◽  
Cell Activation ◽  
Tumor Regression ◽  
Ex Vivo ◽  
Interleukin 2 ◽  
Proliferative Potential

Abstract In a human melanoma model of tumor antigen (TA)–based immunization, we tested the functional status of TA-specific CD8+ cytotoxic T lymphocytes. A “quiescent” phenotype lacking direct ex vivo cytotoxic and proliferative potential was identified that was further characterized by comparing its transcriptional profile to that of TA-specific T cells sensitized in vitro by exposure to the same TA and the T-cell growth factor interleukin 2 (IL-2). Quiescent circulating tumor-specific CD8+ T cells were deficient in expression of genes associated with T-cell activation, proliferation, and effector function. This quiescent status may explain the observed lack of correlation between the presence of circulating immunization-induced lymphocytes and tumor regression. In addition, the activation of TA-specific T cells by in vitro antigen recall and IL-2 suggests that a complete effector phenotype might be reinstated in vivo to fulfill the potential of anticancer vaccine protocols.

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