scholarly journals 323 Immunogenic syngeneic model MC38-OVA for the preclinical evaluation of immune evasion and checkpoint blockade

2021 ◽  
Vol 9 (Suppl 3) ◽  
pp. A348-A348
Author(s):  
Jessie Wang ◽  
Kaixia Lian ◽  
Jia Zheng ◽  
Chenpan Nie ◽  
Annie An ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Tumor Immunity ◽  
Immune Evasion ◽  
Syngeneic Mouse ◽  
Ifn Γ ◽  
Draining Lymph Nodes ◽  
Syngeneic Tumors

BackgroundThe development of immuno-oncology (I/O) therapeutics has revolutionized the cancer treatment landscape. Despite this achievement, the mechanism behind limited responses is poorly understood. Tumor immune evasion has been reported to arise through the loss of tumor necrosis factor (TNF) signaling, interferon-γ (IFN-γ) signaling, and antigen presentation pathways, which are crucial to CD8+ T cell-mediated killing. Syngeneic mouse models have been widely used as they have an intact immune system, are easily accessible, and have a vast array of historical data for comparison. However, limited syngeneic models respond to immune checkpoint inhibitors, possibly due to low intrinsic immunogenicity. The expression of ovalbumin (OVA) has previously shown to sufficiently alter the susceptibility of syngeneic tumors to host T cell-mediated responses. In this study, the newly developed OVA-expressing MC38 syngeneic line was characterized for tumor immunity, checkpoint blockade response and response durability.MethodsMurine colon cancer MC38 cells were transduced by lentiviral vector with chicken OVA coding cDNA. A single clone was selected, and OVA expression was confirmed by western blot. The MC38-OVA cells were subcutaneously implanted into immunocompetent mice to evaluate the tumorigenicity and in vivo response to anti-PD-1 antibody treatment. Blood was collected 2 days post final dose of anti-PD-1 treatment for phenotypic analysis by FACS. Spleen and tumor draining lymph nodes were collected at termination for FACS analysis of IFN-γ+ T cells and OVA specific CD8+ T cells. Adoptive transfer was evaluated by challenge studies in both MC38-OVA and MC38 tumor-bearing mice with T cells derived from MC38-OVA mice, anti-PD-1 cured mice and OT-I mice. In vitro killing assays were performed to evaluate the function of adoptive CD3+ T cells transfer.ResultsOVA-expressing MC38 presented complete regression under anti-PD-1 treatment in vivo. T cell expansion was observed after anti-PD-1 treatment in peripheral blood with increased IFN-γ+ T cells in both tumor-draining lymph nodes and spleen. Additionally, anti-PD-1 cured mice generated robust tumor specific memory T cell, which successfully inhibited MC38-OVA and MC38 tumor growth following adoptive transfer. CD3+ T cells from MC38-OVA-bearing mice and OT-I mice showed anti-tumor immunity in vivo. In vitro killing assay demonstrated increased immunity.ConclusionsSyngeneic mouse tumor models are preferred preclinical models for I/O research, despite limited intrinsic immunogenicity. OVA expression in syngeneic tumors largely increased T cell-mediated immunity to enhance antigen-specific T cell responses during tumorigenesis, providing novel immunogenic models for preclinical immunotherapy evaluation.

scholarly journals Development of a System To Study CD4+-T-Cell Responses to Transgenic Ovalbumin-Expressing Toxoplasma gondii during Toxoplasmosis

2004 ◽  
Vol 72 (12) ◽  
pp. 7240-7246 ◽  
Author(s):  
Marion Pepper ◽  
Florence Dzierszinski ◽  
Amy Crawford ◽  
Christopher A. Hunter ◽  
David Roos
Keyword(s):  
T Cells ◽  
T Cell ◽  
Toxoplasma Gondii ◽  
T Cell Responses ◽  
Cell Receptor ◽  
In Vivo Studies ◽  
Cell Responses ◽  
Ifn Γ

ABSTRACT The study of the immune response to Toxoplasma gondii has provided numerous insights into the role of T cells in resistance to intracellular infections. However, the complexity of this eukaryote pathogen has made it difficult to characterize immunodominant epitopes that would allow the identification of T cells with a known specificity for parasite antigens. As a consequence, analysis of T-cell responses to T. gondii has been based on characterization of the percentage of T cells that express an activated phenotype during infection and on the ability of these cells to produce cytokines in response to complex mixtures of parasite antigens. In order to study specific CD4+ T cells responses to T. gondii, recombinant parasites that express a truncated ovalbumin (OVA) protein, in either a cytosolic or a secreted form, were engineered. In vitro and in vivo studies reveal that transgenic parasites expressing secreted OVA are able to stimulate T-cell receptor-transgenic OVA-specific CD4+ T cells to proliferate, express an activated phenotype, and produce gamma interferon (IFN-γ). Furthermore, the adoptive transfer of OVA-specific T cells into IFN-γ−/− mice provided enhanced protection against infection with the OVA-transgenic (but not parental) parasites. Together, these studies establish the utility of this transgenic system to study CD4+-T-cell responses during toxoplasmosis.

Humulus scandens Exhibits Immunosuppressive Effects in Vitro and in Vivo by Suppressing CD4+ T Cell Activation

2014 ◽  
Vol 42 (04) ◽  
pp. 921-934 ◽  
Author(s):  
Jinjin Feng ◽  
Yingchun Wu ◽  
Yang Yang ◽  
Weiqi Jiang ◽  
Shaoping Hu ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
T Cell Activation ◽  
Cell Activation ◽  
Interferon Γ ◽  
Delayed Type Hypersensitivity ◽  
Ifn Γ ◽  
Humulus Scandens

Humulus scandens, rich in flavonoids, is a traditional Chinese medicine. It is widely used in China to treat tuberculosis, dysentery and chronic colitis. In this study, the major active faction of Humulus scandens (H.S) was prepared. Then, its immunosuppressive effects and underlying mechanisms on T cell activation were investigated in vitro and in vivo. Results showed that H.S significantly inhibited the proliferation of splenocytes induced by concanavalin A, lipopolysaccharides, and mixed-lymphocyte reaction in vitro. Additionally, H.S could dramatically suppress the proliferation and interferon-γ (IFN-γ) production from T cells stimulated by anti-CD3 and anti-CD28. Flow cytometric results confirmed that H.S could suppress the differentiation of IFN-γ-producing type 1 helper T cells (Th1). Furthermore, using ovalbumin immunization-induced T cell reaction and CD4+ T-cell-mediated delayed type hypersensitivity reaction, H.S the immunosuppressive effects of H.S was also demonstrated in vivo. Western blot results showed that H.S could impede the activation of both Erk1/2 and P38 in primary T cells triggered by anti-CD3/28. Collectively, the active fraction of H.S showed promising immunosuppressive activities both in vitro and in vivo.

scholarly journals Apoptotic Cells for the Prevention of Cytokine Release Syndrome (CRS) in CAR T-Cell Therapy

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 1626-1626
Author(s):  
Dror Mevorach ◽  
Veronique Amor ◽  
Yehudith Shabat
Keyword(s):  
T Cells ◽  
Dendritic Cells ◽  
T Cell ◽  
Inflammatory Cytokines ◽  
Apoptotic Cells ◽  
Car T ◽  
Ifn Γ ◽  
Pro Inflammatory Cytokines

Abstract Background: Chimeric antigen receptor (CAR)-modified T cells with specificity against CD19 have demonstrated dramatic promise against highly refractory hematologic malignancies. Clinical responses with complete remission rates as high as 90% have been reported in children and adults with relapsed/refractory acute lymphoblastic leukemia (ALL). However, very significant toxicity has been observed and as many as 30% in average developing severe forms of CRS and possibly related neurotoxicity. CRS is occurring due to large secretion of pro-inflammatory cytokines, mainly from macrophages/monocytes, and resembles macrophage-activating syndrome and hemophagocytosis in response to CAR T-secreting IFN-g and possibly additional cytokines. To better understand the mechanisms leading to CRS and to treat or prevent it, we have developed in vitro and in vivo models of CRS with and without CAR-modified T cells. Early apoptotic cells that have been successfully tested for the prevention of acute GVHD, including in 7 ALL patients, were tested in these models for their effect on cytokines and CAR T cell cytotoxicity. Methods: CD19-expressing HeLa cells were used alone or with co-incubation with human macrophages for in vitro experiments and intraperitoneal experiments. Raji was used in vivo for leukemia induction. LPS and IFN-γ were used to trigger additional cytokine release. CD19-specific CAR-modified cells were used (ProMab) for anti-tumor effect against CD19-bearing cells. Cytotoxicity assay was examined in vivo using 7-AAD with flow cytometry and in vitro by survival curves and analysis of tumor load in bone marrow and liver. CRS occurred spontaneously or in response to LPS and IFN-γ. Mouse IL-10, IL-1β, IL-2, IP-10, IL-4, IL-5, IL-6, IFNα, IL-9, IL-13, IFN-γ, IL-12p70, GM-CSF, TNF-α, MIP-1α, MIP-1β, IL-17A, IL-15/IL-15R, and IL-7, as well as 32 human cytokines were evaluated by Luminex technology using the MAPIX system analyzer (Mereck Millipore) and MILLIPLEX Analyst software (Merek Millipore). Mouse IL-6Rα, MIG (CXCL9), and TGF-β1 were evaluated by Quantikine ELISA (R&D systems). Bone marrow and liver were evaluated using flow cytometry and immunohistochemistry. The IFN-γ effect was evaluated by STAT1 phosphorylation and biological products. Human macrophages and dendritic cells were generated from monocytes. Early apoptotic cells were produced as shown in GVHD clinical trial; at least 50% of cells were annexin V-positive and less than 5% were PI-positive. Results: Apoptotic cells had no negative effect in vitro or in vivo on CAR-modified T cells with specificity against CD19. There were comparable E/T ratios for CAR T in the presence or absence of apoptotic cells in vitro, and comparable survival curves in vivo. On the other hand, significant downregulation (p<0.01) of pro-inflammatory cytokines, including IL-6, IP-10, TNF-a, MIP-1α, MIP-1β, was documented. IFN-γ was not downregulated, but its effect on macrophages and dendritic cells was inhibited at the level of phosphorylated STAT1 and IFN-γ-induced expression of CXCL10 and CXCL9 was reduced. Conclusion: CRS evolves from several factors, including tumor biology, interaction with monocytes/macrophages/dendritic cells, and as a response to the CAR T cell effect and expansion. Apoptotic cells decrease pro-inflammatory cytokines that originate from innate immunity and inhibit the IFN-γ effect on monocyte/macrophages/ dendritic cells without harming IFN-γ levels or CAR-T cytotoxicity. Disclosures Mevorach: Enlivex: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding. Amor:Enlivex: Employment. Shabat:Enlivex: Employment.

The Potential of T Cell Immunoglobulin and Mucin-Domain containing-3 (Tim-3) in Designing Novel Immunotherapy for Bladder Cancer

2021 ◽  
Vol 21 ◽  
Author(s):  
Monireh Mohsenzadegan ◽  
Parizad Bavandpour ◽  
Mohammad Reza Nowroozi ◽  
Erfan Amini ◽  
Masoumeh Kourosh-Arami ◽  
...  
Keyword(s):  
Bladder Cancer ◽  
T Cells ◽  
T Cell ◽  
Tumor Immunity ◽  
Checkpoint Inhibitors ◽  
Number Of Patients ◽  
Anti Cancer ◽  
Domain 3

: Targeting inhibitory receptors on T cells in the tumor sites can promote effective anti-tumor immunity in bladder cancer. Unfortunately, the main dilemma is that a large number of patients remain refractory to CTLA-4, PD-1, and PD-L1 blockade therapies. T-cell immunoglobulin and mucin domain 3 (Tim-3) is an inhibitory receptor expressed on T cells and innate immune cells. Both in vivo and in vitro data from patients with advanced cancers support the role of Tim-3 inhibition in satisfactory anti-tumor immunity. In bladder cancer, the expression level of Tim-3 significantly increases with advanced pathological grade and T stage. Therefore, rationality implies that designing novel monoclonal antibodies reactive with Tim-3 alone or in combination with other checkpoint inhibitors may indicate a favorable response in bladder cancer. Here, we aimed to investigate the possibility of targeting Tim-3 as a novel anti-cancer treatment for bladder cancer.

Inhibition of Graft-Versus-Host Disease (GVHD) by Histone Deacetylase Inhibitor (HDAC) SAHA Leads to Downregulation of STAT1 Activation.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 1311-1311
Author(s):  
Corinna Leng ◽  
Cuiling Li ◽  
Judy Ziegler ◽  
Anna Lokshin ◽  
Suzanne Lentzsch ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Histone Deacetylase ◽  
Graft Versus Host Disease ◽  
Host Disease ◽  
Graft Versus Host ◽  
Tnf Α ◽  
Ifn Γ

Abstract Histone deacetylase (HDAC) inhibitors have been shown to reduce development of graft versus host disease [GVHD] following allogeneic bone marrow transplantation [BMT]. Administration of the HDAC inhibitor suberonylanilide hydroxamic acid [SAHA] resulted in a significantly reduced GVHD-dependent mortality following fully MHC-mismatched allogeneic BMT. Median Survival Time (MST) for vehicle and SAHA-treated mice were 7.5 days and 38 days respectively. However, SAHA treatment did not affect T cell activation nor T cell expansion in vitro and in vivo as determined by MLR assays, phenotypic analysis of donor T cells with regard to expression of the CD25 activation antigen and calculation of donor CD4+ and CD8+ T cell numbers on days +3 and +6 post-BMT. Thus, SAHA treatment was not able to inhibit the strong upregulation of CD25 antigen on CD8+ T cells observed during induction of GVHD on days +3 and +6 post-BMT. We therefore focused on the effects of SAHA treatment on efferent immune effects including cytokine secretion and intracellular signaling events in vitro and in vivo following GVHD induction. SAHA treatment broadly inhibited lipopolysaccharide [LPS] and allo-antigen-induced cytokine/chemokine secretion in vitro like MIP-1-α, IP-10, IFN-γ, TNF-α and IL-6 and led also to a significant decrease in IFN-γ and TNF-α levels in vivo following induction of GVHD. Concomitantly, SAHA treatment inhibited phosphorylation of STAT1 and STAT3 in response to LPS and allo-activation in vitro. Furthermore, analysis of liver tissue and spleens from SAHA-treated animals with GVHD showed a significant decrease in phosphorylated STAT1. In contrast SAHA treatment had only moderate effects on p38 or ERK1,2 Mitogen-activated Protein Kinase (MAPK) pathway underscoring the relevance of the inhibition of the STAT1 pathway. In conclusion, GVHD is associated with a strong induction of phosphorylation of STAT1 in the liver and spleen and SAHA-dependent reduction of GVHD is associated with systemic and local inhibition of pSTAT1 and modulation of the inflammatory cytokine milieu during the efferent immune response.

Quantitative and Functional Alterations of the Gamma Delta T-Cell Subset within Follicular Lymphoma Microenvironment.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 2601-2601
Author(s):  
Sophie de Guibert ◽  
Jean-Baptiste Thibert ◽  
Céline Bonnaventure ◽  
Patricia Ame-Thomas ◽  
Céline Pangault ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Follicular Lymphoma ◽  
Peripheral Blood ◽  
Γδ T Cells ◽  
Γδ T Cell ◽  
Ifn Γ ◽  
Nonpeptide Antigens

Abstract T cells carrying a γδ TCR account for less than 5% of CD3pos T cells in healthy individuals but are key effectors of innate immunity through the recognition of some unprocessed nonpeptide antigens of both self and foreign origin. Whereas the Vδ2 subpopulation represents more than 70% of peripheral blood γδ T cells, the Vδ1 subset is mainly located in the mucosal tissue. Increasing evidence suggest that γδ T cells have potent antitumor activity and are implicated in the defense against some haematological and epithelial malignancies. Moreover, Vδ2 T cells constitute an attractive immunotherapy strategy since they could be expanded and activated both in vivo and in vitro using synthetic phosphoantigens and aminobiphosphonates. Such strategies are currently tested in preliminary clinical trials, notably in follicular lymphoma (FL). However, an exhaustive phenotypic and functional characterisation of γδ T cells in this disease, including tumor infiltration, is still lacking. We first explored the composition of FL microenvironment using a multicolour flow cytometry analysis. We observed a significant decrease in the percentage of myeloid (LinnegCD11cposHLADRpos) and plasmacytoid (LinnegCD123posHLADRpos) dendritic cells (P = .0011 and P &lt; .0001, respectively) in FL compared to normal secondary lymphoid organs. In addition, among CD3pos T cells, the proportion of follicular helper T cells (CD4posCXCR5posICOShi) was increased (P = .001) whereas regulatory T-cell (CD4posCD25posfoxp3pos) frequency was not altered. When considering the γδ T-cell compartment, we first highlighted a reduction of the Vδ2 subset in normal tonsils (Vδ2 = 23.48 ± 0.15% of γδ T cells, n = 11) when compared with peripheral blood. Remaining non-δ2 γδT cells were predominantly δ1 T cells. More importantly, infiltrating γδ T cells were significantly decreased in lymph node biopsies from FL patients (mean = 0.48 ± 0.4% of CD3pos T cells; n = 27) when compared both to normal tonsils (mean = 2.49 ± 1.6% of CD3pos T cells; n = 33) (P &lt; .0001) and reactive lymph nodes (mean = 2.64 ± 2.6% of CD3pos T cells; n = 9) (P = .0009). This reduction affected both the Vδ1 and Vδ2 T-cell subsets. The functionality of γδ T cells was then assessed by the measurement of cell expansion and production of IFN-γ upon stimulation with the isopentenyl pyrophosphate (IPP) phosphoantigen. Amplification rate in vitro reached 14.6 ± 4.6 fold in tonsils (n = 10) but only 4.36 ± 1.9 fold in FL samples (n = 7) (P &lt; .002) after 5 days of culture in the presence of IPP + IL-2 + IL-15. When focusing on the δ2 subset, this difference was further increased with a 40-fold amplification in tonsil and a 3-fold amplification in FL samples (P = .0004). Evaluation of IFN-γ production using ELISPOT assay revealed a high heterogeneity among tumor samples since 1 to 40% of δ2 T cells were able to respond to IPP stimulation (n = 7). Preliminary data argued for an association between the quantity and the functionality of γδ T cells in FL tumors. In conclusion, we reported an alteration of γδ T cell frequency and functionality within FL tumor niche. The next purpose will be to correlate these in vitro defects with in vivo clinical responses to immunotherapy strategies targeting γδ T cells.

Hematopoietic Multipotent Progenitor Cells Mobilized by G-CSF Can Inhibit Gvhd

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2969-2969
Author(s):  
Maud D'Aveni ◽  
Julien Rossignol ◽  
Ruddy Montandon ◽  
Marie Bouillie ◽  
Flora Zavala ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Progenitor Cells ◽  
Mechanisms Of Action ◽  
Activated T Cells ◽  
Hematopoietic Stem ◽  
Allogeneic Hsct ◽  
Ifn Γ

Abstract Abstract 2969 Backgound. Acute graft-versus-host-disease (aGVHD) is a frequent life threatening complication of allogeneic hematopoietic stem cell transplantation (HSCT). Despite the infusion of higher doses of T cells with the use of G-CSF-mobilized HSC grafts, the incidence of aGVHD is not increased. The mechanisms by which G-CSF-mobilized HSC can control GVHD are imperfectly elucidated. We previously described the mobilization of murin hematopoïetic progenitor cells (HPCs) by G-CSF and FLT3 ligand capable of inducing tolerance against autoimmune diabetes in the nude mice (Kared, Immunity 2006). We now show that G-CSF can mobilize murin HPCs with immunoregulatory functions in the allogeneic immune response and describe their mechanisms of action. Methods. Mobilization of HPCs is performed by subcutaneous administration of human recombinant G-CSF at 200μg/kg per day, for 4 consecutive days in the C57BL6 (H-2b) mouse. HPCs are collected in the spleen by FACS sorting according to their phenotype: Lin- Sca1high cKithigh FLT3low CD34+ CD106+ CD127−. In vitro, functions and mechanisms of action were analyzed by co-cultures with i) T cells (from C57BL6) activated by anti-CD28 and -CD3 mAbs or activated by BALB/c (H-2d) allogeneic splenic LPS matured dendritic cells, ii) C57BL6 splenic selected CD4+CD25high T regulatory T cells activated by anti-CD28 and -CD3 mAbs iii) activated antitumor specific CD8 T cells (C57BL6 ovalbumin specific TCR transgenic T cells). These different cultures were performed in the presence or absence of inhibitors of selective cytokines or other regulatory molecules. In vivo, we assessed the effect of donor HPCs on GVHD development by injecting C57BL6 derived HPCs (0.5×106/mouse), splenic T cells (1×106/mouse) and T depleted bone marrow cells (5×106/mouse) into lethally irradiated (8 Gy) Balb/c recipients. Results. In vitro, as compared to controls without HPCs, after 3 days of culture, HPCs: 1) promote the proliferation of natural T regs activated by anti-CD3 and anti-CD28 (>80% at 3 days of culture compared to control <50%), 2) inhibit the proliferation of activated T cells (>80% T cells blocked before 4 divisions as compared to control-T cells alone >80% after 4 divisions- p<0, 001) and 3) induce the apoptosis of activated T cells (30% increased, p=0, 01). The proliferation of T regs was cell contact dependant and required the presence of TGF-b. The inhibition of T cell activation required IFN γ produced by activated T-cells and some contact-dependent stimuli. In such pro-inflammatory conditions, HPCs differentiate after 4 days in myeloid derived suppressor cells (MDSC). These cells could then produce NO in response to IFN γ and suppress the proliferation of activated T cell. However, T cell suppression was not dependant on L-arginine depletion. Induction of apoptosis of T cells was Fas/Fas-L dependant. Although in the presence of HPCs the proliferation of CD8+ T TCR transgenic against the dominant ovalbumin epitope SIINFEKL was reduced, the cytotoxic response against the SIINFEKL-pulsed EL4 cell line was enhanced (cytotoxicity >90% with HPCs versus <90% w/o HPCs, p<0, 001). In addition, HPCs express CCR7 and CD62L, which should allow their migration to the sites of allopriming. In vivo, none of the mice that had received allogeneic HSCT with HPCs developed clinical or histological GVHD signs as compared to 50% of the control allografted mice without HPCs. Conclusion. Hematopoietic progenitor cells acquire an immunosuppressive potential after G-CSF mobilization. These cells can be isolated from mobilized peripheral stem cells and suppress GVHD while possibly preserving the GVL effect. Work is underway in humans to identify and amplify this population ex vivo for potential therapeutic application in allogeneic HSCT. Disclosures: No relevant conflicts of interest to declare.

The Induction of Inhibitory Pathways in Dendritic Cells May Hamper the Efficient Activation of Anti-Leukemia T Cells within Chemotherapy-Induced Immunogenic Cell Death

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 1019-1019
Author(s):  
Darina Ocadlikova ◽  
Mariangela Lecciso ◽  
Elisa Orioli ◽  
Elena De Marchi ◽  
Sabina Sangaletti ◽  
...  
Keyword(s):  
Immune Response ◽  
T Cells ◽  
T Cell ◽  
Cd8 T Cells ◽  
Ex Vivo ◽  
Atp Release ◽  
Inhibitory Pathways ◽  
Ifn Γ

Abstract BACKGROUND: Overall survival of adult acute myeloid leukemia (AML) is still poor due to the lack of novel and effective therapies. In different malignancies including AML, some chemotherapy agents, such as daunorubicin (DNR) but not cytarabine (Ara-C), activate the immune response via the cross-priming of anti-tumor T cells by dendritic cells (DCs). Such process, known as immunogenic cell death (ICD), is characterized by intracellular and pericellular modifications of tumor cells, such as the cell surface translocation of calreticulin (CRT) and heat shock proteins 70/90 (HSPs 70/90), the extracellular release of ATP and pro-inflammatory factor HMGB1. Alongside with ICD, chemotherapy is known to induce inflammatory modifications within the tumor microenvironment, which may also elicit immunosuppressive pathways. In particular, DCs may be driven to acquire tolerogenic features, which may ultimately affect anti-tumor T-cell responses. In this study, we characterize ICD in AML to evaluate the involvement of some DC-related inhibitory pathways, such as the expression of indoleamine-2,3-dioxygenase 1 (IDO1) and the activation of PD-L1/PD-1 axis. METHODS: AML patients were analyzed at diagnosis.Before and after DNR-based chemotherapy, patient-derived T cells were extensively characterized by FACS and analyzed for their capacity to produce IFN-γ in response to autologous blasts. The AML cell line HL-60 and primary AML cells were then exposed, in vitro, to different drugs, including DNR and, as control drug, Ara-C. Dying cells were tested for the surface expression of CRT and HSPs 70/90, the release of HMGB1 and ATP. Functionally, immature DCs generated from healthy donors were pulsed with DNR-treated AML cells. Then, loaded DCs were tested for the expression of maturation-associated markers and of inhibitory pathways, such as IDO1 and PD-L1 and used to stimulate autologous CD3+ T cells. After co-culture, autologous healthy donor T cells were analyzed for IFN-g production, PD-1 expression and Tregs induction. A mouse model was set up to investigate in vivo the mechanism(s) underlying ICD in AML. The murine myelomonocytic leukemia cell line WEHI was transfected with luciferase PmeLUC probe, inoculated subcutaneously into BALB/c mice and used to measure in vivo ATP release after chemotherapy. Tumor-infiltrating T cells and DCs were characterized and correlated with ATP release. RESULTS: DNR treatment induced ICD-related modifications in both AML cell lines and primary blasts, including CRT, HSP70 and HSP90 exposure on cell surface, HMGB1 release from nucleus to cytoplasm and supernatant increase of ATP. Ex vivo, T-cell monitoring of DNR-treated AML patients displayed an increase in leukemia-specific IFN-g-producing CD4+ and CD8+ T cells in 20/28 evaluated patients. However, FACS analysis of CD8+ effector T cells emerging after chemotherapy showed a significant up-regulation of exhaustion marker such as LAG3 and PD-1, which paralleled with their reduced ability to produce active effector molecules, such as perforin and granzyme. Moreover, an increase of circulating Tregs was observed after DNR-based chemotherapy. In vitro, loading of chemotherapy-treated AML cells into DCs resulted not only in the induction of a maturation phenotype, but also in over-expression of inhibitory pathways, such as IDO1 and PD-L1. The silencing of IDO1 increased the capacity of DCs loaded with DNR-treated AML cells to induce leukemia-specific IFN-γ production by CD4+ and CD8+ T cells. In vivo, DNR therapy of mice inoculated with established murine AML cell line resulted in increased ATP release. Similarly to ex vivo and in vitro results, tumor-infiltrating DCs showed an increase in maturation status. Moreover, CD4+ and CD8+ T cells had increased IFN-γ production, but showed an exhausted phenotype. CONCLUSIONS: Our data confirm that chemotherapy-induced ICD may be active in AML and results in increased leukemia-specific T-cell immune response. However, a deep, ex vivo, in vitro and in vivo characterization of chemotherapy-induced T cells demonstrated an exhausted phenotype, which may be the result of the inhibitory pathways induction in DCs, such as IDO and PD-L1. The present data suggest that combination of chemotherapy with inhibitors of IDO1 and PD-L1 may represent an interesting approach to potentiate the immunogenic effect of chemotherapy, thus resulting in increased anti-leukemia immune response. Disclosures Cavo: Janssen-Cilag, Celgene, Amgen, BMS: Honoraria.

scholarly journals Low-Dose Decitabine Augments the Activation and Anti-Tumor Immune Response of IFN-γ+ CD4+ T Cells Through Enhancing IκBα Degradation and NF-κB Activation

2021 ◽  
Vol 9 ◽  
Author(s):  
Xiang Li ◽  
Liang Dong ◽  
Jiejie Liu ◽  
Chunmeng Wang ◽  
Yan Zhang ◽  
...  
Keyword(s):  
Immune Response ◽  
Cell Proliferation ◽  
T Cells ◽  
T Cell ◽  
Low Dose ◽  
T Cell Proliferation ◽  
Ifn Γ ◽  
Iκbα Degradation

BackgroundCD4+ T cells play multiple roles in controlling tumor growth and increasing IFN-γ+ T-helper 1 cell population could promote cell-mediated anti-tumor immune response. We have previously showed that low-dose DNA demethylating agent decitabine therapy promotes CD3+ T-cell proliferation and cytotoxicity; however, direct regulation of purified CD4+ T cells and the underlying mechanisms remain unclear.MethodsThe effects of low-dose decitabine on sorted CD4+ T cells were detected both in vitro and in vivo. The activation, proliferation, intracellular cytokine production and cytolysis activity of CD4+ T cells were analyzed by FACS and DELFIA time-resolved fluorescence assays. In vivo ubiquitination assay was performed to assess protein degradation. Moreover, phosphor-p65 and IκBα levels were detected in sorted CD4+ T cells from solid tumor patients with decitabine-based therapy.ResultsLow-dose decitabine treatment promoted the proliferation and activation of sorted CD4+ T cells, with increased frequency of IFN-γ+ Th1 subset and enhanced cytolytic activity in vitro and in vivo. NF-κB inhibitor, BAY 11-7082, suppressed decitabine-induced CD4+ T cell proliferation and IFN-γ production. In terms of mechanism, low-dose decitabine augmented the expression of E3 ligase β-TrCP, promoted the ubiquitination and degradation of IκBα and resulted in NF-κB activation. Notably, we observed that in vitro low-dose decitabine treatment induced NF-κB activation in CD4+ T cells from patients with a response to decitabine-primed chemotherapy rather than those without a response.ConclusionThese data suggest that low-dose decitabine potentiates CD4+ T cell anti-tumor immunity through enhancing IκBα degradation and therefore NF-κB activation and IFN-γ production.

scholarly journals Tumor ablation plus co-administration of CpG and saponin adjuvants affects IL-1 production and multifunctional T cell numbers in tumor draining lymph nodes

2020 ◽  
Vol 8 (1) ◽  
pp. e000649
Author(s):  
Tonke K Raaijmakers ◽  
Renske J E van den Bijgaart ◽  
Martijn H den Brok ◽  
Melissa Wassink ◽  
Annemarie de Graaf ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Lymph Nodes ◽  
Tumor Ablation ◽  
Polymorphonuclear Neutrophil ◽  
Cell Numbers ◽  
Draining Lymph Nodes

BackgroundTumor ablation techniques, like cryoablation, are successfully used in the clinic to treat tumors. The tumor debris remaining in situ after ablation is a major antigen depot, including neoantigens, which are presented by dendritic cells (DCs) in the draining lymph nodes to induce tumor-specific CD8+T cells. We have previously shown that co-administration of adjuvants is essential to evoke strong in vivo antitumor immunity and the induction of long-term memory. However, which adjuvants most effectively combine with in situ tumor ablation remains unclear.Methods and resultsHere, we show that simultaneous administration of cytidyl guanosyl (CpG) with saponin-based adjuvants following cryoablation affects multifunctional T-cell numbers and interleukin (IL)-1 induced polymorphonuclear neutrophil recruitment in the tumor draining lymph nodes, relative to either adjuvant alone. The combination of CpG and saponin-based adjuvants induces potent DC maturation (mainly CpG-mediated), antigen cross-presentation (mainly saponin-based adjuvant mediated), while excretion of IL-1β by DCs in vitro depends on the presence of both adjuvants. Most strikingly, CpG/saponin-based adjuvant exposed DCs potentiate antigen-specific T-cell proliferation resulting in multipotent T cells with increased capacity to produce interferon (IFN)γ, IL-2 and tumor necrosis factor-α in vitro. Also in vivo the CpG/saponin-based adjuvant combination plus cryoablation increased the numbers of tumor-specific CD8+T cells showing enhanced IFNγ production as compared with single adjuvant treatments.ConclusionsCollectively, these data indicate that co-injection of CpG with saponin-based adjuvants after cryoablation induces an increased amount of tumor-specific multifunctional T cells. The combination of saponin-based adjuvants with toll-like receptor 9 adjuvant CpG in a cryoablative setting therefore represents a promising in situ vaccination strategy.

Sign in / Sign up

Export Citation Format

Share Document