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

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.

Download Full-text

In Vivo Clearance of an Intracellular Bacterium, Francisella tularensis LVS, Is Dependent on the p40 Subunit of Interleukin-12 (IL-12) but Not on IL-12 p70

Infection and Immunity ◽

10.1128/iai.70.4.1936-1948.2002 ◽

2002 ◽

Vol 70 (4) ◽

pp. 1936-1948 ◽

Cited By ~ 95

Author(s):

Karen L. Elkins ◽

Allison Cooper ◽

Susan M. Colombini ◽

Siobhán C. Cowley ◽

Tara L. Kieffer

Keyword(s):

T Cells ◽

Francisella Tularensis ◽

Intracellular Bacterium ◽

Interleukin 12 ◽

Intracellular Growth ◽

Lethal Challenge ◽

Immune Lymphocytes ◽

Ifn Γ

ABSTRACT To determine the role of interleukin-12 (IL-12) in primary and secondary immunity to a model intracellular bacterium, we have comprehensively evaluated infection with Francisella tularensis LVS in three murine models of IL-12 deficiency. Mice lacking the p40 protein of IL-12 (p40 knockout [KO] mice) and mice treated in vivo with neutralizing anti-IL-12 antibodies survived large doses of primary and secondary LVS infection but never cleared bacteria and exhibited a chronic infection. In dramatic contrast, mice lacking the p35 protein (p35 KO mice) of heterodimeric IL-12 readily survived large doses of primary sublethal LVS infection as well as maximal secondary lethal challenge, with only a slight delay in clearance of bacteria. LVS-immune wild-type (WT) lymphocytes produced large amounts of gamma interferon (IFN-γ), but p35 KO and p40 KO lymphocytes produced much less; nonetheless, similar amounts of NO were found in all cultures containing immune lymphocytes, and all immune lymphocytes were equally capable of controlling intracellular growth of LVS in vitro. Purified CD4+ and CD8+ T cells from both WT and p40 KO mice controlled intracellular growth, even though T cells from WT mice produced much more IFN-γ than those from p40 KO mice, and p40 KO T cells did not adopt a Th2 phenotype. Thus, while IL-12 p70 stimulation of IFN-γ production may be important for bacteriostasis, IL-12 p70 is not necessary for appropriate development of LVS-immune T cells that are capable of controlling intracellular bacterial growth and for clearance of primary or secondary LVS infection. Instead, an additional mechanism dependent on the IL-12 p40 protein, either alone or in another complex such as the newly discovered heterodimer IL-23, appears to be responsible for actual clearance of this intracellular bacterium.

Download Full-text

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

Blood ◽

10.1182/blood.v106.11.1311.1311 ◽

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.

Download Full-text

Histone Deacetylase Inhibitors Induce Immuno-Dominant Suppression of Dendritic Cells.

Blood ◽

10.1182/blood.v106.11.456.456 ◽

2005 ◽

Vol 106 (11) ◽

pp. 456-456 ◽

Cited By ~ 1

Author(s):

Pavan Reddy ◽

Yoshinobu Maeda ◽

Raimon Duran-Struuck ◽

Oleg Krijanovski ◽

Charles Dinarello ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Mhc Class Ii ◽

Cd4 T Cells ◽

Hdac Inhibitors ◽

Class Ii ◽

Wild Type ◽

Tnf Α

Abstract We and others have recently demonstrated that suberoylanilide hydroxamic acid (SAHA), a histone deacetylase (HDAC) inhibitor with anti-neoplastic properties, reduces experimental acute graft-versus-host disease (GVHD). We have now investigated the mechanisms of action of two HDAC inhibitors, SAHA and ITF 2357, on allogeneic immune responses. Bone marrow derived dendritic cells (DCs) were preincubated with the HDAC inhibitors at nanomolar concentrations for 16–18 hours and stimulated with lipopolysaccharide (LPS). Pretreatment of DCs caused a significant reduction in the secretion of TNF-α, IL-12p70 and IL-6 compared to the untreated controls (P< 0.005). Similar effects were seen using human peripheral blood mononuclear cell derived DCs. Pre-treatment of both murine and human DCs also significantly reduced their in vitro stimulation of allogeneic T cells as measured by proliferation and IFN-γ production (P<0.01). We determined the in vivo relevance of these observations utilizing a mouse model where the responses of allogeneic donor bm12 T cells depended on the function of injected host B6 DCs would stimulate. Recipient Class-II −/− B6 (H-2b) received 11 Gy on day -1 and were injected with 4–5 x 106 wild type B6 DCs treated with SAHA or with media on days -1 and 0 and then transplanted with 2 x 106 T cells and 5 x 106 TCDBM cells from either syngeneic B6 or allogeneic bm12 donors. SAHA treatment of DCs significantly reduced expansion of allogeneic donor CD4+ T cells on day +7 after BMT compared to controls (P<0.05). SAHA treatment induced a similarly significant reduction in the expansion of CD8+ cells in Class I disparate [bm1→β2M−/−] model. In vitro, SAHA treatment significantly suppressed the expression of CD40 and CD80 but did not alter MHC class II expression. Surprisingly, when mixed with normal DCs at 1:1 ratio, SAHA treated DCs dominantly suppressed allogeneic T cell responses. The regulation of T cell proliferation was not reversible by addition of IL-12, TNF-α, IL-18, anti-IL-10 or anti-TGFβ, either alone or in combination. Suppression of allogeneic responses was contact dependent in trans-well experiments. To address whether the regulation of SAHA treated DCs required contact with T cells, we devised a three cell experiment where SAHA treated DCs lacked the capacity to present antigens to T cells. DCs from B6 MHC Class II deficient (H-2b) were treated with SAHA and co-cultured with wild type B6 (H-2b) DCs along with purified allogeneic BALB/c (H-2d) CD4+ T cells in an MLR. Allogeneic CD4+ T cells proliferated well, demonstrating the regulation to be dependent on contact between SAHA treated DCs and T cells. To address the in vivo relevance of this suppression, we utilized a well characterized [BALB/c →B6] mouse model of acute GVHD. Recipient B6 animals received 11Gy on day -1 and were injected with of 5 million host type SAHA treated or control DCs on days −1, 0, and +2. Mice were transplanted on day 0 with 2 x 106 T cells and 5 x 106 BM from either syngeneic B6 or allogeneic BALB/c donors. Injection of SAHA treated DCs resulted in significantly better survival (60% vs. 10%, P < 0.01) and significantly reduced serum levels of TNF-α, donor T cell expansion and histopathology of GVHD on day +7 after BMT compared to the controls. We conclue that HDAC inhibitors are novel immunomodulators that regulate DC function and might represent a novel strategy to prevent GVHD.

Download Full-text

Brief Ex Vivo Incubation with Fas Ligand Selectively Depletes Alloreactive T Cells and Antigen Presenting Cells from Stem Cell Grafts

Blood ◽

10.1182/blood-2018-99-117920 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 2033-2033

Author(s):

Hilit Levy-Barazany ◽

Liat Pinkas ◽

Galina Rodionov ◽

Nitzan Marelly ◽

Michal Tzadok ◽

...

Keyword(s):

T Cells ◽

Stem Cell ◽

T Cell ◽

Fas Ligand ◽

Ex Vivo ◽

T Cell Subsets ◽

Ifn Γ ◽

Antigen Presenting

Abstract Graft versus host disease (GvHD) proceeds to be the Achilles' heel of hematopoietic stem cell transplantation, with clinicians continue facing a classic conflict: too much GvHD and the patient is at risk for transplant-related mortality and decreased quality of life; too little GvHD and the patient is at increased risk of relapse of their malignant disease. T cells and antigen presenting cells (APCs) are major components of the hematopoietic G-CSF mobilized peripheral blood cells (PBCs) graft. While GvHD is T cell mediated, the APCs are required for the initiation and maintenance of the GvHD. To reduce the risk for GvHD, grafts are sometimes depleted of their T cells, however, while preventing GvHD, the critically important attributes of graft versus leukemia (GvL) effect and engraftment are reduced significantly. Novel strategies that aim to abrogate or ameliorate GvHD, while preserving engraftment and GvL are of great need. A short incubation (2hr) of G-CSF mobilized PBCs with multimeric Fas ligand (i.e. ApoGraft) selectively induces apoptosis in T cell subsets and APCs (Panels A and B), but not in CD34+ progenitor cells (data not shown). FasL treatment preferentially induces apoptosis in mature T cell subsets which express high levels of Fas (CD95), such as T stem cell memory (TSCM), T central memory (TCM), and T effector memory (TEM) cells, as well as the pro-inflammatory T cell subtypes TH1 and TH17 cells, while no apoptotic signal is detected in the non-expressing CD95 naïve T cells (Panel A). The expression of T cells and APCs activation markers; CD25 and HLA-DR, respectively, is significantly reduced following apoptotic challenge in vitro (Panel C), as well as in transplanted mice (data not shown). Furthermore, upon an activation stimulus with anti CD3/CD28 beads in vitro, ApoGraft derived T cells secrete lower levels of IFN-γ, than G-CSF mobilized PBCs derived T cells (Panel D). To gain deeper understanding of the kinetics of GvHD development in vivo, NSG mice were transplanted with ApoGraft or G-CSF mobilized PBCs. Homing, expansion and differentiation of human leukocytes subtypes within the mice bone marrow, spleen and blood, were monitored 3, 7 and 14 days post transplantation. Decreased levels of T and B cells infiltration and expansion were detected in the spleen (Panels E and F), suggesting reduced formation of allo-reactive T cell clones. Reduced proliferation of these cells was associated with lower levels of IFN-γ secreted to the plasma (Panel H) and was in correlation with reduced GvHD and prolonged survival of the ApoGraft transplanted mice (Panel G). Importantly, we have previously demonstrated both in-vitro and in-vivo that ApoGraft has similar GvL and stem cell engraftment capabilities, compared to control G-CSF mobilized PBCs (data not shown). In conclusion, in contrast to conventional T- cell depletion methods, ApoGraft, an ex-vivo FasL-treated graft, affects both the T-cells and APCs, leading to reduced GvHD, while maintaining GvL and engraftment potential (Panel I). ApoGraft is currently being evaluated in a Phase I/II clinical trial (NCT02828878) in subjects with hematologic malignancies undergoing matched related allo-HSCT. Figure. Figure. Disclosures Levy-Barazany: Cellect Biotherapeutics Ltd: Employment. Pinkas:Cellect Biotherapeutics Ltd: Employment. Rodionov:Cellect Biotherapeutics Ltd: Employment. Marelly:Cellect Biotherapeutics Ltd: Employment. Tzadok:Cellect Biotherapeutics Ltd: Employment. Bakimer:Cellect Biotherapeutics Ltd: Employment. Yarkoni:Cellect Biotherapeutics Ltd: Employment. Peled:Cellect Biotherapeutics Ltd: Consultancy. Zuckerman:Cellect Biotherapeutics Ltd: Consultancy.

Download Full-text

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

Blood ◽

10.1182/blood.v128.22.3711.3711 ◽

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.

Download Full-text

696 Brentuximab vedotin, a CD30-directed antibody-drug conjugate, selectively depletes activated Tregs in vitro and in vivo

Journal for ImmunoTherapy of Cancer ◽

10.1136/jitc-2020-sitc2020.0696 ◽

2020 ◽

Vol 8 (Suppl 3) ◽

pp. A738-A738

Author(s):

Bryan Grogan ◽

Reice James ◽

Michelle Ulrich ◽

Shyra Gardai ◽

Ryan Heiser ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Efflux Pump ◽

Apoptotic Cell ◽

T Cell Subsets ◽

Memory Cells ◽

Antibody Drug Conjugate ◽

Selective Depletion

BackgroundRegulatory T cells (Tregs) play an important role in maintaining immune homeostasis, preventing excessive inflammation in normal tissues. In cancer, Tregs hamper anti-tumor immunosurveillance and facilitate immune evasion. Selective targeting of intratumoral Tregs is a potentially promising treatment approach. Orthogonal evaluation of tumor-infiltrating lymphocytes (TILs) in solid tumors in mice and humans have identified CCR8, and several tumor necrosis family receptors (TNFRs), including TNFSFR8 (CD30), as receptors differentially upregulated on intratumoral Tregs compared to normal tissue Tregs and other intratumoral T cells, making these intriguing therapeutic targets.Brentuximab vedotin (BV) is approved for classical Hodgkin lymphoma (cHL) across multiple lines of therapy including frontline use in stage III/IV cHL in combination with doxorubicin, vinblastine, and dacarbazine. BV is also approved for certain CD30-expressing T-cell lymphomas. BV is comprised of a CD30-directed monoclonal antibody conjugated to the highly potent microtubule-disrupting agent monomethyl auristatin E (MMAE).The activity of BV in lymphomas is thought to primarily result from tumor directed intracellular MMAE release, leading to mitotic arrest and apoptotic cell death.The role CD30 plays in normal immune function is unclear, with both costimulatory and proapoptotic roles described. CD30 is transiently upregulated following activation of memory T cells and expression has been linked to highly activated/suppressive IRF4+ effector Tregs.MethodsHere we evaluated the activity of BV on CD30-expressing T cell subsets in vitro and in vivo.ResultsTreatment of enriched T cell subsets with clinically relevant concentrations of BV drove selective depletion of CD30-expressing Tregs > CD30-expressingCD4+ T memory cells, with minimal effects on CD30-expressing CD8+ T memory cells. In a humanized xeno-GVHD model, treatment with BV selectively depleted Tregs resulting in accelerated wasting and robust T cell expansion. The observed differential activity on Tregs is likely attributable to significant increases in CD30 expression and reduced efflux pump activity relative to other T cell subsets. Interestingly, blockade of CD25 signaling prevents CD30 expression on T cell subsets without impacting proliferation, suggesting a link between CD25, the high affinity IL-2 receptor, and CD30 expression.ConclusionsTogether, these data suggest that BV may have an immunomodulatory effect through selective depletion of highly suppressive CD30-expressing Tregs.AcknowledgementsThe authors would like to thank Michael Harrison, PharmD for their assistance in abstract preparation.Ethics ApprovalAnimals studies were approved by and conducted in accordance with Seattle Genetics Institutional Care and Use Committee protocol #SGE-024.

Download Full-text

Gamma Interferon Production, but Not Perforin-Mediated Cytolytic Activity, of T Cells Is Required for Prevention of Toxoplasmic Encephalitis in BALB/c Mice Genetically Resistant to the Disease

Infection and Immunity ◽

10.1128/iai.72.8.4432-4438.2004 ◽

2004 ◽

Vol 72 (8) ◽

pp. 4432-4438 ◽

Cited By ~ 52

Author(s):

Xisheng Wang ◽

Hoil Kang ◽

Takane Kikuchi ◽

Yasuhiro Suzuki

Keyword(s):

T Cells ◽

T Cell ◽

Nude Mice ◽

Genetic Resistance ◽

Cytolytic Activity ◽

Gamma Interferon ◽

Toxoplasmic Encephalitis ◽

Wild Type ◽

Ifn Γ

ABSTRACT We previously showed the requirement of both T cells and gamma interferon (IFN-γ)-producing non-T cells for the genetic resistance of BALB/c mice to the development of toxoplasmic encephalitis (TE). In order to define the role of IFN-γ production and the perforin-mediated cytotoxicity of T cells in this resistance, we obtained immune T cells from spleens of infected IFN-γ knockout (IFN-γ−/−), perforin knockout (PO), and wild-type BALB/c mice and transferred them into infected and sulfadiazine-treated athymic nude mice, which lack T cells but have IFN-γ-producing non-T cells. Control nude mice that had not received any T cells developed severe TE and died after discontinuation of sulfadiazine treatment due to the reactivation of infection. Animals that had received immune T cells from either wild-type or PO mice did not develop TE and survived. In contrast, nude mice that had received immune T cells from IFN-γ−/− mice developed severe TE and died as early as control nude mice. T cells obtained from the spleens of animals that had received either PO or wild-type T cells produced large amounts of IFN-γ after stimulation with Toxoplasma gondii antigens in vitro. In addition, the amounts of IFN-γ mRNA expressed in the brains of PO T-cell recipients did not differ from those in wild-type T-cell recipients. Furthermore, PO mice did not develop TE after infection, and their IFN-γ production was equivalent to or higher than that of wild-type animals. These results indicate that IFN-γ production, but not perforin-mediated cytotoxic activity, by T cells is required for the prevention of TE in genetically resistant BALB/c mice.

Download Full-text

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

Journal for ImmunoTherapy of Cancer ◽

10.1136/jitc-2021-sitc2021.323 ◽

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.

Download Full-text

The Role of Interleukin-10 (IL-10) in IL-15–Mediated T-Cell Responses

Blood ◽

10.1182/blood.v90.11.4513 ◽

1997 ◽

Vol 90 (11) ◽

pp. 4513-4521 ◽

Cited By ~ 24

Author(s):

Dieter Körholz ◽

Ursula Banning ◽

Halvard Bönig ◽

Markus Grewe ◽

Marion Schneider ◽

...

Keyword(s):

Cell Proliferation ◽

T Cells ◽

T Cell ◽

Interleukin 10 ◽

T Cell Proliferation ◽

Cell Production ◽

Cd25 Expression ◽

Tnf Α ◽

Ifn Γ

Abstract Interleukin-15 (IL-15) is a potent T-cell stimulating factor, which has recently been used for pre-clinical in vivo immunotherapy. Here, the IL-15 effect on CD3-stimulated peripheral human T cells was investigated. IL-15 induced a significant T-cell proliferation and upregulated CD25 expression. IL-15 significantly enhanced T-cell production of interferon-γ (IFN-γ), tumor necrosis factor-α (TNF-α), and IL-10. Between 10- and 100-fold greater concentrations of IL-15 were necessary to reach a biological effect equivalent to that of IL-2. Blockade of IL-2 binding to the high-affinity IL-2 receptor did not affect the IL-15 effects, suggesting that IL-15 did not act by inducing endogenous IL-2. Exogenously administered IL-10 significantly reduced the IL-15 and IL-2–mediated IFN-γ and TNF-α production, whereas T-cell proliferation and CD25 expression were not affected. The inhibitory effects of exogenously administered IL-10 on T-cell cytokine production appeared indirect, and are likely secondary to decreased IL-12 production by accessory cells. Inhibition of endogenous IL-10 binding to the IL-10 receptor significantly increased IFN-γ and TNF-α release from T cells. These data suggest that endogenous IL-10 can regulate activated T-cell production of IFN-γ and TNF-α via a paracrine negative feedback loop. The observations of this study could be of relevance for the therapeutic use of IL-15 in vivo.

Download Full-text

Phosphoantigen-activated Vγ2Vδ2 T cells antagonize IL-2–induced CD4+CD25+Foxp3+ T regulatory cells in mycobacterial infection

Blood ◽

10.1182/blood-2008-06-162792 ◽

2009 ◽

Vol 113 (4) ◽

pp. 837-845 ◽

Cited By ~ 55

Author(s):

Guangming Gong ◽

Lingyun Shao ◽

Yunqi Wang ◽

Crystal Y. Chen ◽

Dan Huang ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Treatment Regimen ◽

T Regulatory Cells ◽

Mycobacterial Infection ◽

T Cell Subsets ◽

Regulatory Cells ◽

Ifn Γ ◽

Vγ2vδ2 T Cells

Abstract Although Foxp3+ T regulatory cells (Tregs) are well documented for their ability to suppress various immune cells, T-cell subsets capable of counteracting Tregs have not been demonstrated. Here, we assessed phosphoantigen-activated Vγ2Vδ2 T cells for the ability to interplay with Tregs in the context of mycobacterial infection. A short-term IL-2 treatment regimen induced marked expansion of CD4+CD25+Foxp3+ T cells and subsequent suppression of mycobacterium-driven increases in numbers of Vγ2Vδ2 T cells. Surprisingly, activation of Vγ2Vδ2 T cells by adding phosphoantigen Picostim to the IL-2 treatment regimen down-regulated IL-2–induced expansion of CD4+CD25+Foxp3+ T cells. Consistently, in vitro activation of Vγ2Vδ2 T cells by phosphoantigen plus IL-2 down-regulated IL-2–induced expansion of CD4+CD25+Foxp3+ T cells. Interestingly, anti–IFN-γ–neutralizing antibody, not anti–TGF-β or anti–IL-4, reduced the ability of activated Vγ2Vδ2 T cells to down-regulate Tregs, suggesting that autocrine IFN-γ and its network contributed to Vγ2Vδ2 T cells' antagonizing effects. Furthermore, activation of Vγ2Vδ2 T cells by Picostim plus IL-2 treatment appeared to reverse Treg-driven suppression of immune responses of phosphoantigen-specific IFNγ+ or perforin+ Vγ2Vδ2 T cells and PPD-specific IFNγ+αβ T cells. Thus, phos-phoantigen activation of Vγ2Vδ2 T cells antagonizes IL-2–induced expansion of Tregs and subsequent suppression of Ag-specific antimicrobial T-cell responses in mycobacterial infection.

Download Full-text