scholarly journals Molecular profiles and immunomodulatory activities of glioblastoma-derived exosomes

2020 ◽  
Vol 2 (1) ◽  
Author(s):  
Juliana Hofstatter Azambuja ◽  
Nils Ludwig ◽  
Saigopalakrishna Yerneni ◽  
Aparna Rao ◽  
Elizandra Braganhol ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell Activation ◽  
Cd8 T Cells ◽  
Immune Cells ◽  
Cell Activation ◽  
Immune Cell ◽  
Size Exclusion ◽  
Cell Functions

Abstract Background Glioblastoma is one of the most immunosuppressive human tumors. Emerging data suggest that glioblastoma-derived exosomes (GBex) reprogram the tumor microenvironment into a tumor-promoting milieu by mechanisms that not yet understood. Methods Exosomes were isolated from supernatants of glioblastoma cell lines by size exclusion chromatography. The GBex endosomal origin, size, protein cargos, and ex vivo effects on immune cell functions were determined. GBex were injected intravenously into mice to evaluate their ability to in vivo modulate normal immune cell subsets. Results GBex carried immunosuppressive proteins, including FasL, TRAIL, CTLA-4, CD39, and CD73, but contained few immunostimulatory proteins. GBex co-incubated with primary human immune cells induced simultaneous activation of multiple molecular pathways. In CD8+ T cells, GBex suppressed TNF-α and INF-γ release and mediated apoptosis. GBex suppressed natural killer (NK) and CD4+ T-cell activation. GBex activated the NF-κB pathway in macrophages and promoted their differentiation into M2 cells. Inhibition of the NF-κB pathway in macrophages reversed the GBex-mediated effects. GBex-driven reprogramming of macrophages involved the release of soluble factors that promoted tumor proliferation in vitro. In mice injected with GBex, the frequency of splenic CD8+ T cells, NK cells, and M1-like macrophages was reduced, while that of naïve and M2-like macrophages increased (P < .05). Conclusions GBex reprogrammed functions of all types of immune cells in vitro and altered their frequency in vivo. By creating and sustaining a highly immunosuppressive environment, GBex play a key role in promoting tumor progression.

442 ICT01, an anti-BTN3A mAb that activates Vg9Vd2 T cells, plus interleukin-2: a potent and promising combination for cancer immunotherapy

2020 ◽  
Vol 8 (Suppl 3) ◽  
pp. A468-A468
Author(s):  
Aude de Gassart ◽  
Patrick Brune ◽  
LE Suong ◽  
Sophie Agaugué ◽  
Emmanuel Valentin ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cancer Immunotherapy ◽  
T Cell Activation ◽  
Immune Cells ◽  
Cell Activation ◽  
Human Pbmcs ◽  
Vγ2vδ2 T Cells

Background gdT-cells are attractive targets for cancer immunotherapy given their strong cytolytic and pro-inflammatory cytokine secretion activities, and the association between tumor infiltration and positive prognosis.1 2 ImCheck Therapeutics is developing ICT01, an anti-human butyrophilin-3A (BTN3A/CD277) mAb specifically activating g9d2 T-cells in a phosphoantigen (pAg)-independent manner. ICT01 is currently in a Phase 1/2a study in solid and hematologic tumors (NCT04243499).IL-2 has been shown to expand g9d2 T-cells in vitro and in non-human primates in presence of pAgs.3 4 5 We wanted to characterize the proliferative effects of combining ICT01 with IL-2 on γ9δ2 T-cells as an approach to potentiate g9d2 T-cell mediated cancer immunotherapy.Methods g9d2 T-cell activation and expansion was assessed in vitro in human PBMCs treated with ICT01±IL-2, and in vivo, in the blood of immunocompromised NCG mice engrafted with 20 × 106 human PBMCs and treated with ICT01 (single IV dose, 5 mg/kg on Day 1) ±IL-2 (0.3MIU/kg IP on Day 1–4). A dose-ranging ICT01 (single IV dose, 1 or 5 mg/kg on Day 1)+IL-2 combination (1 MIU SC QD on Days 1–5) study was conducted in cynomolgus monkeys.ResultsIn PBMCs cultures in vitro, ICT01 selectively activated g9d2 T-cells and IL-2 significantly enhanced ICT01-mediated g9d2 T-cell proliferation, this compartment reaching >50% of T-cells after 8 days of treatment versus ~10% with ICT01 alone. This was confirmed in vivo in mice models. Flow cytometry analysis of mice blood revealed a 5.5-fold increase in human g9d2 T-cell number in the combination groups compared to ICT01 or IL-2 alone treated animals, with g9d2 T-cell frequency reaching ~35% of the CD3+ T-cell compartment. In Cynomolgus, a specific expansion and activation of peripheral g9d2 T-cells from ~1–2% at baseline to up to 30% of T cells 7 days post ICT01 administration was observed. No ICT01 effect was observed on other immune cells. Histopathological examinations revealed a trend towards higher numbers of g9d2 T-cells in several organs in ICT01+IL-2 treated monkeys. There was no evidence for a systemic cytokine release syndrome at any time point. Adverse effects with variable severity were observed, most of them being reversible and commonly associated with IL-2 alone, and not reported in the IND-enabling GLP toxicity study with ICT01 monotherapy at doses up to 100 mg/kg.ConclusionsThese results demonstrate the ability of ICT01+IL-2 combination to trigger profound γ9δ2 T-cell activation and expansion, suggesting that the clinical combination of ICT01 with a lymphoproliferative cytokine (e.g., IL-2) may be a novel therapeutic approach for cancer patients.Ethics ApprovalPseudonymized samples isolated from healthy volunteers: whole blood by ImCheck Therapeutics under the agreement n° 7173 between ImCheck Therapeutic SAS and EFS PACA (Etablissement Français du Sang Provence-Alpes-cote d’Azur)ReferencesGentles AJ, Newman AM, Liu CL, et al. The prognostic landscape of genes and infiltrating immune cells across human cancers. Nature Medicine 2015;21(8):938–945.Tosolini M, Pont F, Poupot M, et al. Assessment of tumor-infiltrating TCRVγ9Vδ2 γδ lymphocyte abundance by deconvolution of human cancers microarrays. OncoImmunology 2017;6(3):e1284723.Nada MH, Wang H, Workalemahu G, Tanaka Y, Morita CT. Enhancing adoptive cancer immunotherapy with Vγ2Vδ2 T cells through pulse zoledronate stimulation. Journal for ImmunoTherapy of Cancer 2017;5(1):9.Sicard H, Ingoure S, Luciani B, et al. In Vivo Immunomanipulation of Vγ9Vδ2 T cells with a synthetic phosphoantigen in a preclinical nonhuman primate model. The Journal of Immunology 2005;175(8):5471–5480.Ali Z, Shao L, Halliday L, et al. Prolonged (E)-4-Hydroxy-3-Methyl-But-2-Enyl pyrophosphate-driven antimicrobial and cytotoxic responses of pulmonary and systemic Vγ2Vδ2 T cells in macaques. The Journal of Immunology 2007;179(12):8287–8296.

scholarly journals SAT0108 RESPONSE TO ABATACEPT IS ASSOCIATED WITH THE INHIBITION OF PROTEASOME Β1I EXPRESSION IN T CELLS OF PATIENTS WITH RHEUMATOID ARTHRITIS

2020 ◽  
Vol 79 (Suppl 1) ◽  
pp. 988.2-988
Author(s):  
K. Ghannam ◽  
L. Martinez Gamboa ◽  
C. Kedor ◽  
L. Spengler ◽  
G. R. Burmester ◽  
...  
Keyword(s):  
Rheumatoid Arthritis ◽  
T Cells ◽  
T Cell ◽  
T Cell Activation ◽  
Cd8 T Cells ◽  
Cell Activation ◽  
York Academy ◽  
Cell Functions ◽  
Induction Of Apoptosis

Background:Abatacept (CTLA4-Ig) is a biological DMARD (bDMARD) for treatment of rheumatoid arthritis (RA) and selectively modulates the co-stimulatory signal by CD28: CD80/CD86 interaction required for T cell activation. CD28 mediated signaling regulates many T cell functions including cytokine production. The role of proteasome was approved in many autoimmune diseases, and the effect of Abatacept on modifying cytokines including interferon-γ that alters proteasome proteolytic activities was shown in RA.Objectives:To characterize the effect of Abatacept on the expression and proteolytic activity of the immunoproteasome.Methods:Effects of Abatacept on the proteasome system were investigated in 39 patients with rheumatoid arthritis over a period of 24 weeks. Using real time PCR, transcript expression levels of constitutive and corresponding immunoproteasome catalytic subunits were investigated at baseline (T0), week 16 (T16) and week 24 (T24) in sorted blood cells. Proteasomal activity and induction of apoptosis after proteasome inhibition were also evaluated in cellular subsets.Results:Upon treatment with Abatacept, remission or low disease activity according to DAS28 was achieved in 55 % of patients at T16 and in 70 % at T24. By Two- (time and type of response) way ANOVA, a significant reduction of proteasome immunosubunit β1i expression was shown only in CD4+ and CD8+ T cells of prolonged responders at both T16 and T24 (P= 0.0390 andP= 0.0198, respectively). One-way ANOVA analysis for each response group separately confirmed the results and showed significant reduction at T24;P= 0.0396 difference between T0 and T24,P= 0.0260 between T16 and T24 in CD4+ T cells of the same group. Abatacept did not influence chymotrypsin like activity of proteasome, which is carried out by the subunit β5i and had no effect on induction of apoptosis under exposure to a proteasome inhibitor in-vitro.Conclusion:Treatment with Abatacept showed a clear effect on the expression of the proteasome immunosubunit β1i. This phenomenon was only seen in CD4+ and CD8+ T cells of prolonged responding patients with RA suggesting an association between persistent induction of β1i and failure to the T cell directed therapy with Abatacept.References:[1]Keating, G.M., Abatacept: A Review of its Use in the Management of Rheumatoid Arthritis. Drugs, 2013. 73(10): p. 1095-1119.[2]Ferrington, D.A. and D.S. Gregerson, Immunoproteasomes: Structure, Function, and Antigen Presentation. Progress in molecular biology and translational science, 2012. 109: p. 75-112.[3]Marti, L., et al., Alterations in Cytokine Profile and Dendritic Cells Subsets in Peripheral Blood of Rheumatoid Arthritis Patients before and after Biologic Therapy. Annals of the New York Academy of Sciences, 2009. 1173: p. 334-42.Disclosure of Interests:Khetam Ghannam: None declared, Lorena Martinez Gamboa: None declared, Claudia Kedor Consultant of: Advisory Board for Novartis Pharma GmbH, Lydia Spengler: None declared, Gerd Rüdiger Burmester Consultant of: AbbVie Inc, Eli Lilly, Gilead, Janssen, Merck, Roche, Pfizer, and UCB Pharma, Speakers bureau: AbbVie Inc, Eli Lilly, Gilead, Janssen, Merck, Roche, Pfizer, and UCB Pharma, Eugen Feist Consultant of: Novartis, Roche, Sobi, Lilly, Pfizer, Abbvie, BMS, MSD, Sanofi, Speakers bureau: Novartis, Roche, Sobi, Lilly, Pfizer, Abbvie, BMS, MSD, Sanofi

561 DuoBody®-PD-L1×4–1BB (GEN1046) induces superior immune-cell activation, cytokine production and cytotoxicity by combining PD-L1 blockade with conditional 4–1BB co-stimulation

2020 ◽  
Vol 8 (Suppl 3) ◽  
pp. A595-A595
Author(s):  
Alexander Muik ◽  
Isil Altintas ◽  
Rachelle Kosoff ◽  
Friederike Gieseke ◽  
Kristina Schödel ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
T Cells ◽  
T Cell ◽  
Cd8 T Cells ◽  
Cell Activation ◽  
Cytokine Production ◽  
Immune Cell ◽  
Anti Tumor Activity

BackgroundCheckpoint inhibitors targeting the PD-1/PD-L1 axis (CPI) have changed the treatment paradigm and prognosis for patients with advanced solid tumors; however, many patients experience limited benefit due to treatment resistance. 4-1BB co-stimulation can activate cytotoxic T-cell- and NK-cell-mediated anti-tumor immunity and has been shown to synergize with CPI in preclinical models. DuoBody-PD­L1×4-1BB is a first-in-class, Fc-silenced, bispecific next-generation checkpoint immunotherapy that activates T cells through PD-L1 blockade and simultaneous PD-L1-dependent 4-1BB co-stimulation. Here we present preclinical evidence for the mechanism of action of DuoBody-PD-L1×4-1BB, and proof-of-concept using mouse-reactive mbsAb-PD-L1×4-1BB in vivo.MethodsRNA sequencing analyses was performed on primary human CD8+ T cells that were co-cultured with PD-L1+ monocytes in the presence of anti-CD3/anti-CD28 and test compounds. T-cell proliferation and cytokine production were analyzed in primary human T-cell and mixed lymphocyte reaction (MLR) assays in vitro, and using patient-derived tumor-infiltrating lymphocytes (TILs). Cytotoxic activity was assessed in co-cultures of CLDN6+PD-L1+ MDA-MB-231 tumor cells and CLDN6-TCR+CD8+ T cells. Anti-tumor activity of mbsAb-PD-L1×4-1BB was tested in vivo using the CT26 mouse tumor model. Immunophenotyping of the tumor microenvironment (TME), tumor-draining lymph nodes (tdLNs) and peripheral blood was performed by flow cytometry.ResultsDuoBody-PD-L1×4-1BB significantly induced expression of genes associated with immune cell proliferation, migration and cytokine production in activated CD8+ T cells, which were not altered by CPI. DuoBody-PD-L1×4-1BB dose-dependently enhanced expansion of human TILs ex vivo. DuoBody-PD-L1×4-1BB dose-dependently enhanced T-cell proliferation and pro-inflammatory cytokine production in vitro (e.g. IFNγ and TNFα; in polyclonal and antigen-specific T-cell proliferation assays and MLR), which was dependent on crosslinking to PD-L1+ cells and superior to CPI or the combination of Fc-silenced PD-L1- and 4-1BB-specific antibodies. DuoBody-PD-L1x4-1BB induced upregulation of degranulation marker CD107a and granzyme B in CD8+ T cells, resulting in antigen-specific T-cell-mediated cytotoxicity of MDA-MB-231 tumor cells in vitro, superior to CPI. In mice bearing subcutaneous CT26 tumors, a model that was insensitive to PD-L1 blockade, mbsAb-PD-L1×4-1BB elicited tumor rejection in the majority of the mice at active dose levels and significantly improved survival. Dose-dependent anti-tumor activity was associated with expansion of tumor antigen-specific T cells in the blood and enhanced immune-cell activation in tdLNs and TME.ConclusionsCombining PD-L1 blockade with conditional 4-1BB co-stimulation using bispecific antibodies induced T-cell activation, expansion, and cytotoxic activity in vitro and potent anti-tumor activity in vivo superior to CPI. DuoBody-PD-L1×4-1BB is currently being evaluated in patients with advanced solid tumors in a first-in-human trial (NCT03917381).Ethics ApprovalAll mice studies were performed by BioNTech SE at its research facilities in Germany, and the mice were housed in accordance with German federal and state policies on animal research. All experiments were approved by the regulatory authorities for animal welfare in Germany. The use of tumor tissue resections was approved by BioNTech SE‘s Ethics Board, approval number 837.309.12 (8410-F).

scholarly journals CD8+ T Cell Metabolic Rewiring Defined by Single-Cell RNA-Sequencing Identifies a Critical Role of ASNS Expression Dynamics in T Cell Differentiation

2021 ◽  
Author(s):  
Juan Fernandez-Garcia ◽  
Fabien Franco ◽  
Sweta Parik ◽  
Antonino A Pane ◽  
Dorien Broekaert ◽  
...  
Keyword(s):  
T Cells ◽  
Cell Differentiation ◽  
T Cell ◽  
T Cell Activation ◽  
Cd8 T Cells ◽  
Cell Activation ◽  
Cd8 T Cell ◽  
T Cell Differentiation

Cytotoxic T cells dynamically rewire their metabolism during the course of an immune response. While T cell metabolism has been extensively studied at phenotypic endpoints of activation and differentiation, the underlying dynamics remain largely elusive. Here, we leverage on single-cell RNA-sequencing (scRNA-seq) measurements of in vitro activated and differentiated CD8+ T cells cultured in physiological media to resolve these metabolic dynamics. We find that our scRNA-seq analysis identifies most metabolic changes previously defined in in vivo experiments, such as a rewiring from an oxidative to an anabolism-promoting metabolic program during activation to an effector state, which is later reverted upon memory polarization. Importantly, our scRNA-seq data further provide a dynamic description of these changes. In this sense, our data predict a differential time-dependent reliance of CD8+ T cells on the synthesis versus uptake of various non-essential amino acids during T cell activation, which we corroborate with additional functional in vitro experiments. We further exploit our scRNA-seq data to identify metabolic genes that could potentially dictate the outcome of T cell differentiation, by ranking them based on their expression dynamics. Among the highest-ranked hits, we find asparagine synthetase (Asns), whose expression sharply peaks for effector CD8+ T cells and further decays towards memory polarization. We then confirm that these in vitro Asns expression dynamics are representative of an in vivo situation in a mouse model of viral infection. Moreover, we find that disrupting these expression dynamics in vitro, by depleting asparagine from the culture media, delays central-memory polarization. Accordingly, we find that preventing the decay of ASNS by stable overexpression at the protein level in vivo leads to a significant increase in effector CD8+ T cell expansion, and a concomitant decrease in central-memory formation, in a mouse model of viral infection. This shows that ASNS expression dynamics dictate the fate of CD8+ T cell differentiation. In conclusion, we provide a resource of dynamic expression changes during CD8+ T cell activation and differentiation that is expected to increase our understanding of the dynamic metabolic requirements of T cells progressing along the immune response cascade.

scholarly journals Response to abatacept is associated with the inhibition of proteasome β1i expression in T cells of patients with rheumatoid arthritis

RMD Open ◽  
2020 ◽  
Vol 6 (3) ◽  
pp. e001248
Author(s):  
Khetam Ghannam ◽  
Lorena Martinez Gamboa ◽  
Claudia Kedor ◽  
Lydia Spengler ◽  
Ulrike Kuckelkorn ◽  
...  
Keyword(s):  
Rheumatoid Arthritis ◽  
T Cells ◽  
T Cell ◽  
Disease Activity ◽  
T Cell Activation ◽  
Cd8 T Cells ◽  
Cell Activation ◽  
Cell Functions ◽  
Induction Of Apoptosis

ObjectiveAbatacept is a biological disease-modifying antirheumatic drug (DMARD) used for the treatment of rheumatoid arthritis (RA) and modulates the costimulatory signal by cluster of differentiation (CD)28:CD80/CD86 interaction required for T cell activation. Since CD28-mediated signalling regulates many T cell functions including cytokine production of, for example, interferons (IFNs), it is of interest to clarify, whether response to abatacept has an effect on the IFN inducible immunoproteasome, as a central regulator of the immune response.MethodsEffects of abatacept on the proteasome were investigated in 39 patients with RA over a period of 24 weeks. Using real-time PCR, transcript levels of constitutive and corresponding immunoproteasome catalytic subunits were investigated at baseline (T0), week 16 (T16) and week 24 (T24) in sorted blood cells. Proteasomal activity and induction of apoptosis after proteasome inhibition were also evaluated.ResultsAbatacept achieved remission or low disease activity in 55% of patients at T16 and in 70% of patients at T24. By two-way analysis of variance (ANOVA), a significant reduction of proteasome immunosubunit β1i was shown only in CD4+ and CD8+ T cells of sustained responders at both T16 and T24. One-way ANOVA analysis for each response group confirmed the results and showed a significant reduction at T24 in CD4+ and CD8+ T cells of the same group. Abatacept did not influence chymotrypsin-like activity of proteasome and had no effect on induction of apoptosis under exposure to a proteasome inhibitor in vitro.ConclusionThe reduction of proteasome immunosubunit β1i in T cells of patients with RA with sustained response to abatacept suggests association of the immunoproteasome of T cells with RA disease activity.

scholarly journals 781 Validation of the combinatorial effect of blinatumomab and nivolumab in cancer therapy

2020 ◽  
Vol 8 (Suppl 3) ◽  
pp. A831-A831
Author(s):  
Tienan Wang ◽  
Qing Lin ◽  
Jie Zhang
Keyword(s):  
T Cells ◽  
T Cell ◽  
T Cell Activation ◽  
Cd8 T Cells ◽  
Immune Checkpoint ◽  
Culture System ◽  
Cell Activation ◽  
Checkpoint Inhibitors

BackgroundCancer immunotherapies, including immune checkpoint inhibitors, CAR-T, cancer vaccines and bispecific antibodies, have been brought to spot light in recent years as several therapeutic strategies targeting the immune system have produced exciting clinical results. Bispecific antibody typically play dual roles in blocking the immune checkpoint and redirecting/re-boosting the function of the immune effector cells. Blinatumomab belongs to CD3 bispecific T cell engager (CD3 BiTE), which was engineered to harbor two arms binding with CD3 and CD19 simultaneously and direct CD8+ T cells to specifically recognize CD19 positive lymphoma cells to execute cytotoxicity. Approval of Blinatumomab for patients with relapse/refractory B cell acute lymphoblastic leukemia (ALL) has driven remarkable increase in combination studies of Blinatumomab with other immunotherapies such as checkpoint inhibitors.MethodsIn this study, we developed CD8+ T cytotoxic system targeting different B lymphoma cell line and fully validated the function of Blinatumomab in promoting target tumor cell lysis by primary CD8+ T cells (figure 1). In addition, we established a mixed lymphocyte and tumor system to mimic physiological TME to dissect the combinational role of Nivolumab and Blinatumomab (figure 2).ResultsThe result suggest that combinatory therapy is highly depend on the dosage of Blinatumomab and also T cell number in the TME, which might give an instruction for ongoing clinical trial design. Finally, we have employed humanized mouse models bearing Raji or Daudi tumor cells to further validate this combination treatment in vivo. Both In-vivo and In-vitro data support that Blinatumomab is dominant in activing T cell and Nivolumab can only exhibit synergistic effect under suboptimal dosage of Blinatumomab.Abstract 781 Figure 1Establishment of In vitro co-culture system for CD3 BiTEestablish in vitro human PBMC based system to validate CD3 BiTE functionAbstract 781 Figure 2Opdivo and CD3 BiTE CombinationOpdivo could further promote T cell activation under the treatment of CD3 BiTEConclusionsSuccessfully establish in vitro system to evaluate the function of CD3 BiTE and also take advantage of MLR/tumor co-culture system to demonstrate PD1 antibody could further promote T cell activation under appropriate dosage of CD3 BiTE.

Modulation of T-Cell Functions in KIR2DL3 (CD158b) Transgenic Mice

Blood ◽  
1999 ◽  
Vol 94 (7) ◽  
pp. 2396-2402 ◽  
Author(s):  
Anna Cambiaggi ◽  
Sylvie Darche ◽  
Sophie Guia ◽  
Philippe Kourilsky ◽  
Jean-Pierre Abastado ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Transgenic Mice ◽  
T Cell Activation ◽  
Cell Activation ◽  
Killer Cell ◽  
In Vitro Proliferation ◽  
Cell Functions ◽  
Double Transgenic Mice

In humans, a minor subset of T cells express killer cell Ig-like receptors (KIRs) at their surface. In vitro data obtained with KIR+ β and γδ T-cell clones showed that engagement of KIR molecules can extinguish T-cell activation signals induced via the CD3/T-cell receptor (TCR) complex. We analyzed the T-cell compartment in mice transgenic for KIR2DL3 (Tg-KIR2DL3), an inhibitory receptor for HLA-Cw3. As expected, mixed lymphocyte reaction and anti-CD3 monoclonal antibody (MoAb)-redirected cytotoxicity exerted by freshly isolated splenocytes can be inhibited by engagement of transgenic KIR2DL3 molecules. In contrast, antigen and anti-CD3 MoAb-induced cytotoxicity exerted by alloreactive cytotoxic T lymphocytes cannot be inhibited by KIR2DL3 engagement. In double transgenic mice, Tg-KIR2DL3 × Tg-HLA-Cw3, no alteration of thymic differentiation could be documented. Immunization of double transgenic mice with Hen egg white lysozime (HEL) or Pigeon Cytochrome-C (PCC) was indistinguishable from immunization of control mice, as judged by recall antigen-induced in vitro proliferation and TCR repertoire analysis. These results indicate that KIR effect on T cells varies upon cell activation stage and show unexpected complexity in the biological function of KIRs in vivo.

scholarly journals In Vivo Role of Dendritic Cells in a Murine Model of Pulmonary Cryptococcosis

2006 ◽  
Vol 74 (7) ◽  
pp. 3817-3824 ◽  
Author(s):  
Karen L. Wozniak ◽  
Jatin M. Vyas ◽  
Stuart M. Levitz
Keyword(s):  
T Cells ◽  
Dendritic Cells ◽  
T Cell Activation ◽  
Cell Activation ◽  
Ex Vivo ◽  
Interleukin 2 ◽  
Mouse Lung

ABSTRACT Dendritic cells (DC) have been shown to phagocytose and kill Cryptococcus neoformans in vitro and are believed to be important for inducing protective immunity against this organism. Exposure to C. neoformans occurs mainly by inhalation, and in this study we examined the in vivo interactions of C. neoformans with DC in the lung. Fluorescently labeled live C. neoformans and heat-killed C. neoformans were administered intranasally to C57BL/6 mice. At specific times postinoculation, mice were sacrificed, and lungs were removed. Single-cell suspensions of lung cells were prepared, stained, and analyzed by microscopy and flow cytometry. Within 2 h postinoculation, fluorescently labeled C. neoformans had been internalized by DC, macrophages, and neutrophils in the mouse lung. Additionally, lung DC from mice infected for 7 days showed increased expression of the maturation markers CD80, CD86, and major histocompatibility complex class II. Finally, ex vivo incubation of lung DC from infected mice with Cryptococcus-specific T cells resulted in increased interleukin-2 production compared to the production by DC from naïve mice, suggesting that there was antigen-specific T-cell activation. This study demonstrated that DC in the lung are capable of phagocytosing Cryptococcus in vivo and presenting antigen to C. neoformans-specific T cells ex vivo, suggesting that these cells have roles in innate and adaptive pulmonary defenses against cryptococcosis.

scholarly journals TRPM2 Is Not Required for T-Cell Activation and Differentiation

2022 ◽  
Vol 12 ◽  
Author(s):  
Niels C. Lory ◽  
Mikolaj Nawrocki ◽  
Martina Corazza ◽  
Joanna Schmid ◽  
Valéa Schumacher ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
T Cell Activation ◽  
Cell Activation ◽  
Transient Receptor Potential ◽  
Cell Function ◽  
Intestinal Inflammation ◽  
Activation Markers

Antigen recognition by the T-cell receptor induces a cytosolic Ca2+ signal that is crucial for T-cell function. The Ca2+ channel TRPM2 (transient receptor potential cation channel subfamily M member 2) has been shown to facilitate influx of extracellular Ca2+ through the plasma membrane of T cells. Therefore, it was suggested that TRPM2 is involved in T-cell activation and differentiation. However, these results are largely derived from in vitro studies using T-cell lines and non-physiologic means of TRPM2 activation. Thus, the relevance of TRPM2-mediated Ca2+ signaling in T cells remains unclear. Here, we use TRPM2-deficient mice to investigate the function of TRPM2 in T-cell activation and differentiation. In response to TCR stimulation in vitro, Trpm2-/- and WT CD4+ and CD8+ T cells similarly upregulated the early activation markers NUR77, IRF4, and CD69. We also observed regular proliferation of Trpm2-/- CD8+ T cells and unimpaired differentiation of CD4+ T cells into Th1, Th17, and Treg cells under specific polarizing conditions. In vivo, Trpm2-/- and WT CD8+ T cells showed equal specific responses to Listeria monocytogenes after infection of WT and Trpm2-/- mice and after transfer of WT and Trpm2-/- CD8+ T cells into infected recipients. CD4+ T-cell responses were investigated in the model of anti-CD3 mAb-induced intestinal inflammation, which allows analysis of Th1, Th17, Treg, and Tr1-cell differentiation. Here again, we detected similar responses of WT and Trpm2-/- CD4+ T cells. In conclusion, our results argue against a major function of TRPM2 in T-cell activation and differentiation.

scholarly journals 708 Novel ways to exploit IL-21 to augment adoptive T cell transfer therapy against tumors

2021 ◽  
Vol 9 (Suppl 3) ◽  
pp. A737-A737
Author(s):  
Anna Cole ◽  
Guillermo Rangel RIvera ◽  
Aubrey Smith ◽  
Megan Wyatt ◽  
Brandon Ware ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cd8 T Cells ◽  
Immune Cells ◽  
Striking Difference ◽  
T Cell Therapy ◽  
Cell Immunity ◽  
Emory University

BackgroundIL-21 enhances the anti-tumor capacity of adoptively transferred CD8+ T cells, while IL-2 and IL-15 impair T cell immunity by driving their expansion to a more differentiated status. Yet, these cytokines can act on many different immune cells. Given the potency of IL-21, we tested if this cytokine directly augments T cells or rather if it enhances other immune cells in the culture that indirectly improves T cell therapy.MethodsTo test this question, splenocytes from pmel-1 transgenic mice were used, as all CD8+ T cells express a transgenic TCR specific for tumor-antigen gp10025–33 overexpressed on melanoma. We then peptide activated naïve CD8+ T cells enriched or not from the spleen of pmel-1 mice and expanded them in the presence of IL-21 or IL-2 (10 ng/mL) for four days. Expanded pmel-1 from these various cultures were then restimulated with irradiated splenocytes pulsed with gp10025–33 and grown an additional seven days with IL-2 (10 ng/mL), irrespective of their initial cytokine condition. The in vitro memory phenotype, exhaustion profile, and cytokine secretion of these cultures were then assayed. Furthermore, mice bearing B16KVP melanoma tumors were infused with pmel-1 T cells expanded via these various approaches and compared for their relative capacity to engraft, persist, and regress tumor in vivo.ResultsInterestingly, we discovered that IL-21-treated T cells generated from bulk splenocytes are phenotypically and functionally distinct from IL-21-treated isolated T cells. Upon restimulation, IL-21-treated T cells from bulk splenocytes exhibited an exhausted phenotype that was like anergic IL-2-treated T cells. Moreover, few cells expressed CD62L but expressed heightened markers of suppression, including TIM3, PD-1, and EOMES. Moreover, they produced more effector molecules, including granzyme B and IFN-gamma. In vivo IL-21-treated T cells expanded from bulk splenocytes engrafted and persisted poorly, in turn mediating suboptimal regression of melanoma. Conversely, IL-21 dramatically bolstered the engraftment and antitumor activity of T cells only if they were first isolated from the spleen prior to their expansion and infusion into the animal.ConclusionsCollectively, our data shows that IL-21 may improve ACT therapy best when used directly on antitumor CD8+ T cells. Further studies will illuminate the mechanism behind this striking difference and determine whether other cell subsets reactive to IL-21 cause T cell dysfunction and/or reduced bioavailability. These findings are important for defining the best culture conditions in which to use IL-21 for ACT.AcknowledgementsWe would like to acknowledge Emory University, The Winship Cancer Institute, and the Pediatrics/Winship Flow Cytometry Core.Ethics ApprovalAll animal procedures were approved by the Institutional Animal Care and Use Committee of Emory University, protocol number 201900225.

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