scholarly journals Chemotherapy-Dependent ATP Release from Leukemia Dying Cells Induces Indoleamine 2,3-Dioxygenase 1 in Dendritic Cells

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 3711-3711
Author(s):  
Mariangela Lecciso ◽  
Darina Ocadlikova ◽  
Elena De Marchi ◽  
Elisa Orioli ◽  
Sabina Sangaletti ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cd8 T Cells ◽  
Atp Release ◽  
Cell Immune Response ◽  
Tnf Α ◽  
Ido1 Expression ◽  
Ifn Γ

Abstract BACKGROUND: Overall survival of adult acute myeloid leukemia (AML) remains poor due to the lack of novel and effective therapies. The cancer cell death induced by some chemotherapeutic agents, especially anthracyclines, such as daunorubicin (DNR), named immunogenic cell death (ICD), is characterized by intra- and peri-cellular modifications, which favor the induction of anti-tumor T-cell immune response. Among them, the extracellular release of adenosine triphosphate (extracellular ATP, eATP) from dying tumor cells primes dendritic cells (DCs) by activating purinergic P2X7 receptors, thus eliciting the presentation of tumor antigens to T cell. DCs are key regulators of adaptive immunity, promoting or suppressing T-cell responses. One of the suppressive mechanisms involves the expression of indoleamine 2,3-dioxygenase 1 (IDO1), which plays a major role in the induction of T-cell tolerance through the expansion of regulatory T cells (Tregs). The present study aimed at evaluating the involvement of IDO-1 during ATP-driven ICD in AML. METHODS: AML patients were analyzed at diagnosis and after DNR-based chemotherapy. Ex vivo T cells were characterized by FACS and tested for their capacity to produce IFN-γ in response to autologous blasts. Then, CD8+IFN-γ-producing T cells were expanded and further characterized. ATP was used as an ICD representative model. In vitro, murine WEHI-3B and human HL-60 leukemic cell lines and primary blasts were tested for ATP release after DNR treatment. To in vivo investigate DNR-induced ICD, WEHI-3B cells stable transfected with luciferase PmeLUC were inoculated subcutaneously in BALB/c mice to measure ATP release directly from tumor mass. Tumor infiltrating DCs and T cells were characterized by FACS and immunohistochemistry after chemotherapy and plasma levels of cytokines were measured. In vitro DNR-treated AML cells were pulsed into immature DCs, previously generated from healthy donors. DCs maturation and IDO1 expression were examined (by FACS and western blot, respectively) and correlated with the presence of ATP in culture medium. IDO-driven Tregs induction was assessed. Finally, functional immunological tests were performed in vitro to test the ability of Tregs to inhibit leukemia antigen-specific IFN-γ production (FACS analysis) by ICD-activated T cells. RESULTS: After chemotherapy, 15/23 AML patients had an increase in leukemia-specific IFN-γ producing CD4+ and CD8+ T cells. Also an increase of Tregs was observed with a peak at day 21. CD8+ IFN-γ-producing T cells, which resulted in a skewing toward an effector memory phenotype, were activated and cytotoxic against autologous AML blasts but showed features of exhaustion and were defective in perforin production. In vitro and in vivo DNR induced ATP release from AML cells. In vivo the analysis of tumor-infiltrating T cells after treatment has shown an exhausted phenotype of cytotoxic CD8+ cells, increased IFN-γ+ Tregs and decreased TNF-α+ effector T cells. DNR treatment also increased in vivo plasma levels of cytokines IFN-γ, IL-1β, TNF-α, IL-12. Moreover, in DNR-treated mice we observed a significant increase of CD11c+ mature DCs which express IDO1 in tumor infiltrate. In vitro, loading of DNR-treated AML cells into DCs resulted in increased maturation, but also in IDO1 induction. Interestingly, extracellular ATP was directly involved in DCs maturation and IDO1 expression via purinergic receptor P2Y11. ICD-driven DCs were able to expand Tregs in an IDO-dependent manner. Finally, ICD both triggers a leukemia-specific IFN-γ production by CD8+T cells and induces Tregs, via IDO1-expressing DCs, which in turn inhibit leukemia-specific T cell. CONCLUSIONS: Overall, our data indicate that in AML chemotherapy-induced ICD has contrasting, and not fully elucidated, effects on T-cell immune response, resulting in the induction of leukemia-specific CTLs, albeit with defective features, and Tregs. In this scenario, the effects of ATP release from dying leukemia cells on DCs may be pivotal, as indicated by its capacity to concomitantly induce DC maturation and activation as well as tolerogenic function via IDO1. The combination of novel immunological drugs, such as IDO1 and/or checkpoint inhibitors, with conventional chemotherapy may represent an interesting approach to contrast tolerance induction and, then, fully exploit the immunogenic effect of chemotherapy. Disclosures Martinelli: Genentech: Consultancy; Amgen: Consultancy, Speakers Bureau; Celgene: Consultancy, Speakers Bureau; Novartis: Speakers Bureau; Ariad: Consultancy, Speakers Bureau; Roche: Consultancy, Speakers Bureau; Pfizer: Consultancy, Speakers Bureau; MSD: Consultancy; BMS: Speakers Bureau. Cavo:Celgene: Consultancy, Honoraria; Millennium: Consultancy, Honoraria; Bristol-Myers Squibb: Consultancy, Honoraria; Janssen-Cilag: Consultancy, Honoraria; Amgen: Consultancy, Honoraria.

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.

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.

Monocytic Myeloid-Derived Suppress Cells Restore Adipogenic Bone Marrow Microenvironment in Aplastic Anemia By Inhibiting Intra-BM CD8+ T Cells Proliferation

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 1211-1211
Author(s):  
Ying Qu ◽  
Zhengxu Sun ◽  
Yan Yuan ◽  
Fen Wang ◽  
Kunpeng Wu ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Cell Proliferation ◽  
T Cells ◽  
T Cell ◽  
Cd8 T Cells ◽  
T Cell Proliferation ◽  
Immune Balance ◽  
Ifn Γ

Aplastic anemia (AA) is a hematopoietic disorder resulted from immune-related hypocellular hematopoiesis in bone marrow (BM). It has been clearly addressed that the activated T cells contribute to the exhaustion of hematopoietic progenitors and hypo-hematopoiesis. The adipogenic BM is one of the characteristics to make AA diagnosis. However, little is known about the relationship of intra-BM immune imbalance and hematopoietic microenvironment abnormity in this disease entity. Functional hematopoiesis relies on not only abundant hematopoietic stem cells (HSCs) but also the balanced supportive hematopoietic niche. Intra-BM immune balance, at either cellular or cytokine level, is one of the key footstones to maintain hematopoietic microenvironment. Various intra-BM immune cellular components play both sides of one coin. Among them, myeloid-derived suppressive cells (MDSCs) are heterogeneous myeloid progenitor cells characterized by the negative immune response in cancers and other inflammatory diseases. In BM aspiration and biopsy samples from the patients who were diagnosed as AA in our study, massive activated lymphocytes infiltration and adipocytes accumulation were observed. Interestingly, the absolute numbers of immune modulatory MDSCs either in AA patients' PB or in BM of immune-related AA mice were reduced, indicating a potential link between polarized BM adipo-osteogenic microenvironment and immune disorder under AA circumstance. We thus adopted AA mice model to look into the embedded details both in vivo and in vitro. We clarified that BM components were more vulnerable to the attack of CD8+ T cells than that of CD4+ T cells. Taking into the fact that BM adipocytes are more abundant either in AA patients or in AA mice models, we differentiated mesenchymal stromal cells (MSCs), the major BM stroma cells, into osteoblastic or adipogenic lineages to mimic the osteo-adipogenic differentiation in BM microenvironment. Interestingly, CD8+ T cells and interferon-γ(IFN-γ) exerted dramatically adipocytic stimulation on BM-MSCs either in vitro or in vivo, by determination of increasing expression of adipogenetic genes including Ap2, Perilipin, Pparg and Cebpα, as well as staining of Oil Red O and perilipin. To dissect intra-BM cellular immune balance, MDSCs were isolated as representative immune regulating population to investigate their function on osteo-adipogenic balance. Interestingly, not CD11b+Ly6G+Ly6C-granulocytic-MDSCs (gMDSCs) but CD11b+Ly6G-Ly6C+monocytic-MDSCs (mMDSCs) inhibited both T cell proliferation and IFN-γ production. Addition of L-NMMA, the antagonist of iNOS pathway in mMDSCs-containing system restored T cell proliferative curve and cell numbers, whereas Nor-NOHA, the antagonist of Arg-1 pathway didn't abrogate mMDSCs' immune-regulation properties, indicating that mMDSCs inhibited T cell proliferation via iNOS pathway. We then performed single dose or multi-dose injection of mMDSCs in AA mice to see whether mMDSCs are able to reconstitute the impacted hematopoiesis. Single injection of mMDSCs was able to prevent from CTL infiltration in a very short term. However, multi-injection of mMDSCs showed significant benefit in overall survival rate compared to AA mice. We further detected the function of mMDSCs on polarized BM-MSCs adipo-osteogenic differentiation potential. To detect sequential BM adipogenetic progression in AA microenvironment, we performed in vivo fluorescent microscopy on AP2 (Fabp4)-Cre×mT/mG reporting mice at different transfusion time points of T cells and mMDSCs. GFP-expressing AP2+ adipocytes accumulated adjacently to perivascular niches whose boarders were labelled by Dextran-CY5 in a time-dependent manner after T cell infusion. Monocytic MDSCs transfused AA mice showed decreased GFP+ adipocytes which was coincident with our in vitro findings. In conclusion, intra-BM immune balance is one of the environmental factors seesawing by activating and suppressive ends to support functional hematopoiesis. Adoptive transfusion of mMDSCs, the immune-suppressive population might be a novel immune-regulating strategy to treat AA, relying on not only restoring the intra-BM immune balance but also improving stroma's multi-differentiating microenvironment. Figure Disclosures No relevant conflicts of interest to declare.

scholarly journals Multiple T Cell Subsets Control Francisella tularensis LVS Intracellular Growth Without Stimulation Through Macrophage Interferon γ Receptors

2003 ◽  
Vol 198 (3) ◽  
pp. 379-389 ◽  
Author(s):  
Siobhán C. Cowley ◽  
Karen L. Elkins
Keyword(s):  
T Cells ◽  
T Cell ◽  
Francisella Tularensis ◽  
T Cell Subsets ◽  
Wild Type ◽  
Intracellular Growth ◽  
Tnf Α ◽  
Ifn Γ

A variety of data suggest that in vivo production of interferon (IFN)-γ is necessary, but not sufficient, for expression of secondary protective immunity against intracellular pathogens. To discover specific IFN-γ–independent T cell mediated mechanisms, we took advantage of an in vitro culture system that models in vivo immune responses to the intracellular bacterium Francisella tularensis live vaccine strain (LVS). LVS-immune lymphocytes specifically controlled 99% of the growth of LVS in wild-type murine bone marrow–derived macrophages. Surprisingly, LVS-immune lymphocytes also inhibited LVS intracellular growth by as much as 95% in macrophages derived from IFN-γ receptor knockout (IFNγR KO) mice. CD8+ T cells, and to a lesser degree CD4+ T cells, controlled LVS intracellular growth in both wild-type and IFNγR KO macrophages. Further, a unique population of Thy1+αβ+CD4−CD8− cells that was previously suggested to operate during secondary immunity to LVS in vivo strongly controlled LVS intracellular growth in vitro. A large proportion of the inhibition of LVS intracellular growth in IFNγR KO macrophages by all three T cell subsets could be attributed to tumor necrosis factor (TNF) α. Thus, T cell mechanisms exist that control LVS intracellular growth without acting through the IFN-γ receptor; such control is due in large part to TNF-α, and is partially mediated by a unique double negative T cell subpopulation.

scholarly journals CD28 Costimulation Is Required for In Vivo Induction of Peripheral Tolerance in CD8 T Cells

2002 ◽  
Vol 197 (1) ◽  
pp. 19-26 ◽  
Author(s):  
Melanie S. Vacchio ◽  
Richard J. Hodes
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cd8 T Cells ◽  
Tolerance Induction ◽  
Peripheral Tolerance ◽  
Cd8 Cells ◽  
Costimulatory Signal ◽  
Cd28 Costimulation

Whereas ligation of CD28 is known to provide a critical costimulatory signal for activation of CD4 T cells, the requirement for CD28 as a costimulatory signal during activation of CD8 cells is less well defined. Even less is known about the involvement of CD28 signals during peripheral tolerance induction in CD8 T cells. In this study, comparison of T cell responses from CD28-deficient and CD28 wild-type H-Y–specific T cell receptor transgenic mice reveals that CD8 cells can proliferate, secrete cytokines, and generate cytotoxic T lymphocytes efficiently in the absence of CD28 costimulation in vitro. Surprisingly, using pregnancy as a model to study the H-Y–specific response of maternal T cells in the presence or absence of CD28 costimulation in vivo, it was found that peripheral tolerance does not occur in CD28KO pregnants in contrast to the partial clonal deletion and hyporesponsiveness of remaining T cells observed in CD28WT pregnants. These data demonstrate for the first time that CD28 is critical for tolerance induction of CD8 T cells, contrasting markedly with CD28 independence of in vitro activation, and suggest that the role of CD28/B7 interactions in peripheral tolerance of CD8 T cells may differ significantly from that of CD4 T cells.

scholarly journals DNAM-1 promotes activation of cytotoxic lymphocytes by nonprofessional antigen-presenting cells and tumors

2008 ◽  
Vol 205 (13) ◽  
pp. 2965-2973 ◽  
Author(s):  
Susan Gilfillan ◽  
Christopher J. Chan ◽  
Marina Cella ◽  
Nicole M. Haynes ◽  
Aaron S. Rapaport ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Nk Cells ◽  
Adhesion Molecules ◽  
Cd8 T Cells ◽  
Nk Cell ◽  
Cd8 T Cell ◽  
Antigen Presenting

Natural killer (NK) cells and CD8 T cells require adhesion molecules for migration, activation, expansion, differentiation, and effector functions. DNAX accessory molecule 1 (DNAM-1), an adhesion molecule belonging to the immunoglobulin superfamily, promotes many of these functions in vitro. However, because NK cells and CD8 T cells express multiple adhesion molecules, it is unclear whether DNAM-1 has a unique function or is effectively redundant in vivo. To address this question, we generated mice lacking DNAM-1 and evaluated DNAM-1–deficient CD8 T cell and NK cell function in vitro and in vivo. Our results demonstrate that CD8 T cells require DNAM-1 for co-stimulation when recognizing antigen presented by nonprofessional antigen-presenting cells; in contrast, DNAM-1 is dispensable when dendritic cells present the antigen. Similarly, NK cells require DNAM-1 for the elimination of tumor cells that are comparatively resistant to NK cell–mediated cytotoxicity caused by the paucity of other NK cell–activating ligands. We conclude that DNAM-1 serves to extend the range of target cells that can activate CD8 T cell and NK cells and, hence, may be essential for immunosurveillance against tumors and/or viruses that evade recognition by other activating or accessory molecules.

scholarly journals The Immunomodulatory Effect of Triptolide on Mesenchymal Stem Cells in Vitro

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 5011-5011
Author(s):  
Haiping He ◽  
Atsuko Takahashi ◽  
Yuki Yamamoto ◽  
Akiko Hori ◽  
Yuta Miharu ◽  
...  
Keyword(s):  
Stem Cells ◽  
T Cells ◽  
Cd8 T Cells ◽  
Research Funding ◽  
Complete Medium ◽  
Surface Markers ◽  
Rt Pcr ◽  
Tnf Α ◽  
Ifn Γ

Background: Mesenchymal stromal cells (MSC) are known to have the immunosuppressive ability and have been applied in clinic to treat acute graft-versus-host disease (GVHD), as one of severe complications after hematopoietic stem cells transplantation (HSCT) in Japan. However, MSC are activated to suppress the immune system only upon the stimulation of inflammatory cytokines and the clinical results of MSC therapies for acute GVHD are varied. It is ideal that MSC are primed to be activated and ready to suppress the immunity (=priming) before administration in vivo. Triptolide (TPL) is a diterpene triepoxide purified from a Chinese herb - Tripterygium Wilfordii Hook F (TWHF). It has been shown to possess anti-inflammatory and immunosuppressive properties in vitro. In this study, we aim to use TPL as the activator for umbilical cord-derived MSC (UC-MSC) to entry stronger immunosuppressive status. Methods: The proliferation of UC-MSC with TPL at the indicated concentrations for different time of 24, 48, 72, and 96 hours. Cell counting kit-8(CCK-8) was added in the culture medium to detect cell toxicity and the absorbance was measured using microplate reader. Flow cytometry was used to identify the MSC surface markers expression. TPL-primed UC-MSC were once replaced with fresh medium and co-culture with mixed lymphocyte reaction (MLR) consisted with mononuclear cells (MNCs) stained with CFSE and irradiated allogenic dendritic cell line (PMDC05) in RPMI 1640 medium supplemented with 10 % FBS (complete medium). IDO-1, SOD1, and TGF-β gene expression in TPL-primed UC-MSC and UC-MSC induced by 10 ng/ml IFN-γ and/or 15 ng/ml TNF-α were evaluated by RT-PCR. PDL1 and PDL2 expression in TPL-primed UC-MSC and UC-MSC in response to IFN-γ and/or TNF-α were checked by Flowjo. Results: Exposure of TPL for UC-MSC for 72hour at the concentration above 0.1 μM resulted in the cell damage significantly. Therefore, we added TPL in UC-MSC at 0.01μM of TPL for up to 48 hours, then washed thourouphly for the following culture for experiments. To evaluate the influence of TPL on the surface markers of UC-MSC, we cultured UC-MSC for 4 hours in complete medium following culture with 0.01μM of TPL for 20 hours (TPL-primed UC-MSC). TPL-primed UC-MSC revealed positive for CD105, CD73, and CD90, negative for CD45, CD34, CD14 or CD11b, CD79α or CD19 and HLA-DR surface molecules as same as the non-primed UC-MSC. In MLR suppression by UC-MSC, the TPL-primed UC-MSC activity revealed stronger anti-proliferative effect on the CD4+ and CD8+ T cells activated by allogeneic DC than those of non-primed UC-MSC in MLR. Furthermore, the TPL-primed UC-MSC promoted the expression of IDO-1, SOD1 and TGF-β in response to IFN-γ+/-TNF-α by RT-PCR and enhanced the expression of PD-L1 by FACS analysis. Discussion:In this study, we found the TPL-primed UC-MSC showed stronger antiproliferative potency on CD4+ and CD8+ T cells compared with non-primed UC-MSC. TPL-primed UC-MSC promoted the expression of IDO-1, SOD1 and TGF-β stimulated by IFN-γ+/-TNF-α, although TPL alone did not induce these factors. Furthermore, we found that the PD1 ligand (PD-L1) was induced in TPL-primed UC-MSC, likely IFN-γ enhanced the PD-L1 expression, evaluated by flowcytometry. These results suggested that TPL-primed UC-MSC seemed more sensitive to be activated as the immunosuppressant. Here, we firstly report the new function of TPL to induce the upregulation of immunosuppressive effect, although the mechanisms of TPL inhibition to MSC need to be explore. Conclusively, TPL-primed UC-MSC might be applied for the immunosuppressive inducer of MSC. Figure Disclosures He: SASAGAWA Medical Scholarship: Research Funding; IMSUT Joint Research Project: Research Funding. Nagamura:AMED: Research Funding. Tojo:AMED: Research Funding; Torii Pharmaceutical: Research Funding. Nagamura-Inoue:AMED: Research Funding.

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.

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 Transcription factor Etv6 regulates functional differentiation of cross-presenting classical dendritic cells

2018 ◽  
Vol 215 (9) ◽  
pp. 2265-2278 ◽  
Author(s):  
Colleen M. Lau ◽  
Ioanna Tiniakou ◽  
Oriana A. Perez ◽  
Margaret E. Kirkling ◽  
George S. Yap ◽  
...  
Keyword(s):  
T Cells ◽  
Dendritic Cells ◽  
Transcription Factor ◽  
T Cell ◽  
Cd8 T Cells ◽  
Functional Differentiation ◽  
Specific Gene ◽  
Hematopoietic Stem

An IRF8-dependent subset of conventional dendritic cells (cDCs), termed cDC1, effectively cross-primes CD8+ T cells and facilitates tumor-specific T cell responses. Etv6 is an ETS family transcription factor that controls hematopoietic stem and progenitor cell (HSPC) function and thrombopoiesis. We report that like HSPCs, cDCs express Etv6, but not its antagonist, ETS1, whereas interferon-producing plasmacytoid dendritic cells (pDCs) express both factors. Deletion of Etv6 in the bone marrow impaired the generation of cDC1-like cells in vitro and abolished the expression of signature marker CD8α on cDC1 in vivo. Moreover, Etv6-deficient primary cDC1 showed a partial reduction of cDC-specific and cDC1-specific gene expression and chromatin signatures and an aberrant up-regulation of pDC-specific signatures. Accordingly, DC-specific Etv6 deletion impaired CD8+ T cell cross-priming and the generation of tumor antigen–specific CD8+ T cells. Thus, Etv6 optimizes the resolution of cDC1 and pDC expression programs and the functional fitness of cDC1, thereby facilitating T cell cross-priming and tumor-specific responses.

Sign in / Sign up

Export Citation Format

Share Document