IRAK1-regulated IFN-γ signaling induces MDSC to facilitate immune evasion in FGFR1-driven hematological malignancies

Abstract Background Stem Cell leukemia/lymphoma syndrome (SCLL) presents as a myeloproliferative disease which can progress to acute myeloid leukemia and is associated with the coincident development of B-cell and T-cell lymphomas. SCLL is driven by the constitutive activation of fibroblast growth factor receptor-1 (FGFR1) as a result of chromosome translocations with poor outcome. Mouse models have been developed which faithfully recapitulate the human disease and have been used to characterize the molecular genetic events that are associated with development and progression of the disease. Methods CRISPR/Cas9 approaches were used to generate SCLL cells null for Interleukin receptor associated kinase 1 (IRAK1) and interferon gamma (IFNG) which were introduced into syngeneic hosts through tail vein injection. Development of the disease and changes in immune cell composition and activity were monitored using flow cytometry. Bead-based immunoassays were used to compare the cytokine and chemokine profiles of control and knock out (KO) cells. Antibody mediated, targeted depletion of T cell and MDSCs were performed to evaluate their role in antitumor immune responses. Results In SCLL, FGFR1 activation silences miR-146b-5p through DNMT1-mediated promoter methylation, which derepresses the downstream target IRAK1. IRAK1 KO SCLL cells were xenografted into immunocompetent syngeneic mice where the typical rapid progression of disease was lost and the mice remained disease free. IRAK1 in this system has no effect on cell cycle progression or apoptosis and robust growth of the KO cells in immunodeficient mice suggested an effect on immune surveillance. Depletion of T-cells in immunocompetent mice restored leukemogenesis of the KO cells, and tumor killing assays confirmed the role of T cells in tumor clearance. Analysis of the immune cell profile in mice transplanted with the IRAK1 expressing mock control (MC) cells shows that there is an increase in levels of myeloid-derived suppressor cells (MDSCs) with a concomitant decrease in CD4+/CD8+ T-cell levels. MDSC suppression assays and depletion experiments showed that these MDSCs were responsible for suppression of the T cell mediated leukemia cell elimination. Immuno-profiling of a panel of secreted cytokines and chemokines showed that activation of IFN-γ is specifically impaired in the KO cells. In vitro and in vivo expression assays and engraftment with interferon gamma receptor-1 (IFNGR1) null mice and IFNG KO SCLL cells, showed the leukemia cells produced IFN-γ directly participating in the induction of MDSCs to establish immune evasion. Inhibition of IRAK1 using pacritinib suppresses leukemogenesis with impaired induction of MDSCs and attenuated suppression of CD4+/CD8+ T-cells. Conclusions IRAK1 orchestrates a previously unknown FGFR1-directed immune escape mechanism in SCLL, through induction of MDSCs via regulation of IFN-γ signaling from leukemia cells, and targeting IRAK1 may provide a means of suppressing tumor growth in this syndrome by restoring immune surveillance.

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

Patterns for RANTES Secretion and Intercellular Adhesion Molecule 1 Expression Mediate Transepithelial T Cell Traffic Based on Analyses In Vitro and In Vivo

Journal of Experimental Medicine ◽

10.1084/jem.187.12.1927 ◽

1998 ◽

Vol 187 (12) ◽

pp. 1927-1940 ◽

Cited By ~ 66

Author(s):

Masahiko Taguchi ◽

Deepak Sampath ◽

Takeharu Koga ◽

Mario Castro ◽

Dwight C. Look ◽

...

Keyword(s):

T Cells ◽

Cell Adhesion ◽

T Cell ◽

Cell Surface ◽

Immune Cell ◽

Intercellular Adhesion Molecule ◽

Airway Epithelial ◽

Intercellular Adhesion Molecule 1 ◽

Ifn Γ

Immune cell migration into and through mucosal barrier sites in general and airway sites in particular is a critical feature of immune and inflammatory responses, but the determinants of transepithelial (unlike transendothelial) immune cell traffic are poorly defined. Accordingly, we used primary culture airway epithelial cells and peripheral blood mononuclear cells to develop a cell monolayer system that allows for apical-to-basal and basal-to-apical T cell transmigration that can be monitored with quantitative immunofluorescence flow cytometry. In this system, T cell adhesion and subsequent transmigration were blocked in both directions by monoclonal antibodies (mAbs) against lymphocyte function-associated antigen 1 (LFA-1) or intercellular adhesion molecule 1 (ICAM-1) (induced by interferon γ [IFN-γ] treatment of epithelial cells). The total number of adherent plus transmigrated T cells was also similar in both directions, and this pattern fit with uniform presentation of ICAM-1 along the apical and basolateral cell surfaces. However, the relative number of transmigrated to adherent T cells (i.e., the efficiency of transmigration) was increased in the basal-to-apical relative to the apical-to-basal direction, so an additional mechanism was needed to mediate directional movement towards the apical surface. Screening for epithelial-derived β-chemokines indicated that IFN-γ treatment caused selective expression of RANTES (regulated upon activation, normal T cell expressed and secreted), and the functional significance of this finding was demonstrated by inhibition of epithelial–T cell adhesion and transepithelial migration by anti-RANTES mAbs. In addition, we found that epithelial (but not endothelial) cells preferentially secreted RANTES through the apical cell surface thereby establishing a chemical gradient for chemotaxis across the epithelium to a site where they may be retained by high levels of RANTES and apical ICAM-1. These patterns for epithelial presentation of ICAM-1 and secretion of RANTES appear preserved in airway epithelial tissue studied either ex vivo with expression induced by IFN-γ treatment or in vivo with endogenous expression induced by inflammatory disease (i.e., asthma). Taken together, the results define how the patterns for uniform presentation of ICAM-1 along the cell surface and specific apical sorting of RANTES may serve to mediate the level and directionality of T cell traffic through epithelium (distinct from endothelium) and provide a basis for how this process is precisely coordinated to route immune cells to the mucosal surface and maintain them there under normal and stimulated conditions.

Download Full-text

Donor Immunization with WT1 Peptide Augments Anti-Leukemic Activity After MHC-Matched Bone Marrow Transplantation

Blood ◽

10.1182/blood.v118.21.1896.1896 ◽

2011 ◽

Vol 118 (21) ◽

pp. 1896-1896

Author(s):

Holbrook E Kohrt ◽

Antonia MS Mueller ◽

Jeanette B Baker ◽

Matthew J Goldstein ◽

Evan Newell ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Cd8 T Cells ◽

Tumor Antigen ◽

Cd4 T Cell ◽

Leukemia Cells ◽

Peptide Vaccination ◽

Tumor Bearing ◽

Ifn Γ ◽

Anti Tumor Activity

Abstract Abstract 1896 The curative potential of MHC-matched allogeneic bone marrow transplantation (BMT) is in part due to immunologic graft-versus-tumor (GvT) reactions mediated by donor T cells that recognize host minor histocompatibility antigens. Immunization with leukemia-associated antigens, such as Wilm's Tumor 1 (WT1) peptides, induces a T cell population that is tumor antigen specific. We determined whether BMT combined with immunotherapy using WT1 peptide vaccination of donors induced more potent anti-tumor activity when combined with allotransplantation. WT1 peptide vaccinations of healthy syngeneic or allogeneic donor mice with a 9-mer WT1 peptide (amino acids 126–134, the WT1 9-mer which has the highest binding affinity for H-2Db) and Incomplete Freund's Adjuvant induced CD8+ T cells that were specifically reactive to WT1-expressing FBL3 leukemia cells. We found that compared to vaccination with IFA alone, four weekly WT1 vaccinations induced an increased percentage of WT1-tetramer+CD8 T-cells (0.15% vs. 1%) in the peripheral blood 28 days following the first vaccination (Figure A *p<.001). CD8 T-cells producing IFN-γ+ after co-culture with tumor cells were similarly increased (0.11% vs. 13.6%) at this timepoint (Figure B *p<.001). They were CD44hi suggesting a memory phenotype, specifically reactive to WT1-expressing tumor (FBL3 and not H11), and increased in a vaccination dose-dependent fashion (Figure A and B). Four weekly WT1 vaccinations prevented tumor growth in donors following intravenous leukemia challenge. In contrast, in tumor-bearing mice, WT1 vaccinations failed to induce WT1-tetramer+ or IFN-γ+ CD8 T-cells and were ineffective as a therapeutic vaccine based on intensity of bioluminescence from luciferase-labeled FBL3 leukemia and mortality. BMT from WT1 vaccinated MHC-matched donors including LP/J and C3H.SW, but not C57BL/6 syngeneic donors, into C57BL/6 recipient tumor-bearing mice was effective as a therapeutic maneuver and resulted in eradication of luciferase-labeled FBL3 leukemia and survival of 70–90% of mice. Interestingly, the transfer of total CD8+ T cells from immunized donors was more effective than the transfer of WT1-tetramer+CD8+ T cells, likely as a result of alloreactive and tumor-antigen reactive T cells contained with the donor total CD8+ T cells. Total and tetramer+CD8+ T cells required CD4+ T cell help for maximal anti-tumor activity, which was equivalent in efficacy from immunized or unimmunized CD4+ T cell donors. Total CD4+ T cells, alone, from immunized donors provided no anti-tumor activity. The infused donor LP/J or C3H.SW CD8+ T cells collected from cured C57BL/6 recipients, were highly reactive against WT1-expressing FBL3 leukemia cells (14% IFN-γ+) compared to non-WT1-expressing H11 leukemia cells (5% IFN-γ+). The circulating, WT1-tetramer+CD8+ T cell population expanded in cured recipients, peaking at 3.5% on day 50 and contracting through day 100 post-BMT to 0.56%. These findings show that peptide vaccination of donor mice with a tumor antigen dramatically enhances GvT activity and is synergistic with allogeneic BMT. This novel and broadly applicable approach, using leukemia-associated antigen immunization to enhance GvT by creating an “educated” donor T cell graft for allogeneic transplantation of patients with acute myeloid leukemia and myelodysplastic syndrome, is currently being translated to a Phase 1 clinical trial at our institution. Disclosures: No relevant conflicts of interest to declare.

Download Full-text

Tpl2 Promotes Innate Cell Recruitment and Effector T Cell Differentiation To Limit Citrobacter rodentium Burden and Dissemination

Infection and Immunity ◽

10.1128/iai.00193-17 ◽

2017 ◽

Vol 85 (10) ◽

Cited By ~ 1

Author(s):

Nicole V. Acuff ◽

Xin Li ◽

Krishna Latha ◽

Tamas Nagy ◽

Wendy T. Watford

Keyword(s):

T Cells ◽

T Cell ◽

Cell Function ◽

Immune Cell ◽

Cd4 T Cell ◽

Citrobacter Rodentium ◽

Wild Type ◽

Content Type ◽

Cell Recruitment ◽

Ifn Γ

ABSTRACT Tumor progression locus 2 (Tpl2) is a serine-threonine kinase that regulates Th1 differentiation, secretion of the inflammatory cytokine gamma interferon (IFN-γ), and host defense against the intracellular pathogens Toxoplasma gondii, Listeria monocytogenes, and Mycobacterium tuberculosis. However, relatively little is known about the contribution of Tpl2 to Th17 differentiation and immune cell function during infection with an extracellular pathogen. The goal of this study was to determine whether Tpl2 influences the immune response generated to the extracellular bacterium Citrobacter rodentium, which induces a mixed Th1 and Th17 response. During peak infection with C. rodentium, Tpl2 −/− mice experienced greater bacterial burdens with evidence of dissemination to the liver and spleen but ultimately cleared the bacteria within 3 weeks postinfection, similar to the findings for wild-type mice. Tpl2 −/− mice also recruited fewer neutrophils and monocytes to the colon during peak infection, which correlated with increased bacterial burdens. In mixed bone marrow chimeras, Tpl2 was shown to play a T cell-intrinsic role in promoting both IFN-γ and interleukin-17A production during infection with C. rodentium. However, upon CD4 T cell transfer into Rag −/− mice, Tpl2 −/− CD4 T cells were as protective as wild-type CD4 T cells against the dissemination of bacteria and mortality. These data indicate that the enhanced bacterial burdens in Tpl2 −/− mice are not caused primarily by impairments in CD4 T cell function but result from defects in innate immune cell recruitment and function.

Download Full-text

Leukemia Derived Dendritic Cells (LDCs) Are Functionally Deficient and Inferior to DC/Leukemia Fusion Cells as a Tumor Vaccine for AML.

Blood ◽

10.1182/blood.v106.11.2788.2788 ◽

2005 ◽

Vol 106 (11) ◽

pp. 2788-2788

Author(s):

Jacalyn Rosenblatt ◽

Richard Stone ◽

Corrine Lenahan ◽

Zekwui Wu ◽

Baldev Vasir ◽

...

Keyword(s):

T Cells ◽

Dendritic Cells ◽

T Cell ◽

Interferon Gamma ◽

Tumor Immunity ◽

Peripheral Blood ◽

Tumor Antigens ◽

Tumor Vaccine ◽

Costimulatory Molecules ◽

Leukemia Cells

Abstract We have previously demonstrated that dendritic cells (DCs) fused with malignant cells stimulate anti-tumor immunity by presenting a braod array of tumor antigens in the context of DC mediated costimulation. DCs differentiated from leukemia cells (LDCs) are also being explored as cancer vaccines in which leukemia associated antigens are presented. We examined the phenotypic and functional characteristics of DC/Leukemia fusions and LDCs to assess their potential as tumor vaccines. Leukemia blasts were isolated from peripheral blood of patients with AML. CD34 selection was performed on a subset of samples by magnetic bead separation. LDCs were generated by culturing blasts in the presence of GM-CSF, IL-4 and TNFα for 7 days. Alternatively, leukemia cells were fused with DC by coculture in the presence of polyethylene glycol. Differentiation of leukemic blasts into LDCs resulted in increased expression of HLA-DR and CD 11c. Unlike normal peripheral blood mononuclear cells, differentiation of leukemic blasts resulted in only modest expression of the costimulatory molecules, CD80 and CD86 (mean expression 12% and 30%) and no increase in expression of the maturation marker, CD83 (mean expression 4%). In addition, expression of the leukemia associated antigen c-kit (CD117) was lower on LDCs than on blasts (mean expression 34% on blasts, 15% on LDCs). To assess the capacity of the primitive leukemia clonal population to differentiate into DCs, CD34+ cells were isolated from the blast population and assessed after cytokine differentiation. Cytokine differentiation did not result in upregulation of CD80, CD83, or CD86 expression in the CD34+ population (mean expression 5%, 2%, 17%). In contrast, differentiation of the CD34- population resulted in moderate expression of CD80, CD83 and CD86 (mean expression 15%, 14%, 48%). In contrast to LDCs which do not strongly express co-stimulatory molecules and lose expression of leukemia associated antigens, fusion cells expressed both DC and tumor associated antigens (mean fusion efficiency 27%). The functional characteristics of DC derived from leukemic blasts were examined. Allogeneic T cell proliferation in response to stimulation by LDCs was not significantly higher than after stimulation with undifferentiated blasts (ratio 10:1, mean SI 17% with LDCs vs 9% with undifferentiated blasts, p=0.19). Neither stimulation with blasts nor with LDCs induced T cell production of interferon gamma. In contrast, interferon gamma production by T cells in response to stimulation with fusion cells was higher than after stimulation with undifferentiated blasts. In summary, LDCs do not demonstrate normal upregulation of costimulatory molecules, and lose expression of tumor antigens. In contrast, DC/leukemia fusions coexpress tumor and DC associated markers. While LDCs stimulate interferon gamma production by T cells poorly, fusion cells more potently stimulate interferon gamma production by allogeneic T cells than do undifferentiated blasts. This suggests that LDC may be ineffective as a tumor vaccine in AML, and that fusion cells may be superior to LDC in generating effective anti-tumor immune responses. Strategies to enhance the ability of both LDC and of fusion cells to stimulate anti-tumor immunity are being explored.

Download Full-text

IL-15 Induced Polyclonal CTL Generated From Expanded CBT Cells Against Leukemia Cell Lines Constitutes IFN-γ Producing Cells and TCRγδ Cells

Blood ◽

10.1182/blood.v120.21.1910.1910 ◽

2012 ◽

Vol 120 (21) ◽

pp. 1910-1910

Author(s):

Jeyaraj Antony ◽

Xiaohua Chen ◽

Paul Szabolcs

Keyword(s):

T Cells ◽

Myeloid Leukemia ◽

Target Recognition ◽

Leukemia Cell ◽

Leukemia Cells ◽

Specific Lysis ◽

Target Ratio ◽

Ifn Γ ◽

Ctl Activity ◽

Effector Target

Abstract Abstract 1910 Background: We have previously demonstrated that ex vivo expanded CBT cells expanded with CD3/CD28 co-stimulatory beads + IL-2 and IL-7 were receptive to subsequent in vitro priming against killed lymphoid and myeloid leukemia cells in the presence of IL-7, IL-12, and IL-15, (Davis et al. Cancer Research 2010;70(13):5249). Hypothesis and Objectives: Here, we wanted to test 1) the minimum prerequisite cytokine requirement, hypothesizing that higher specificity may be obtained if less exogenous cytokine is employed. 2) Determine the mechanism of CTL's kill by employing blocking antibodies against HLA-ABC, HLA-DR and TCRγδ recognition sites. 3) Characterize the critical cellular phenotype of the CTL to identify whether the cytotoxicity is specific to CD4, CD8, TCRαβ, or TCRγδcell populations or the observed cytotoxicity is the contribution from multiple cellular phenotypes? Methods: CTL was generated from already expanded >98% pure T cells either with a combination of cytokines (IL-7, -12 & -15) versus IL-15, versus IL-7 alone and were compared from the same cultures by matched pair Student T-test. After 3 weeks of CTL cultures, the cytotoxicity (specific lysis) was tested against fresh IM9 leukemia cells (loaded with BATDA) at an Effector: Target ratio of 40:1, 20:1 and 10:1. The blocking with HLA class I, class II and TCRγδ antibodies were done before performing the CTL assay. ELISPOT assay tested the specificity (non-specific target recognition) and magnitude of activity as measured by IFN-γ spot forming cells (SFC). Results: From the data shown in Figure 1, it is evident that IL-15 alone is sufficient to support both priming and expansion of IM-9 specific CTL. After 3 weeks of CTL cultures, the observed specific lysis of fresh IM9 leukemia with IL-15 alone versus in combination was 73% and 75% respectively at an Effector: Target ratio of 40:1. The generated CTL exhibit no cytotoxicity against non-specific targets against a myeloid leukemia cell line (U937). While cytotoxicity by Europium release assay was comparable, CTL from IL-15 alone cultures displayed higher specificity in ELISPOT assays with much less non-specific target recognition as measured by IFN-γ spot forming cells (SFC) (Figure 2A). Though the relative (fold) expansion of CTL observed in the group with all cytokines is significantly higher, the numbers are comparable between the combination of cytokines and IL-15 alone towards the later weeks of priming and expansion, data not shown. By blocking experiments, it is evident that, even though no single MoAb completely abolished cytotoxicity, the most significant diminution was identified when the effectors were blocked with TCRγδ (Figure 2B). Multicolor FACS analysis indicates that after exposing the CTLs to specific targets, ∼7–8% of the cells are secreting IFN- γ and ∼ 20–25% of the generated CTLs are TCRγδ positive. Immunoscope analysis of TCRγδ spectratype identifies oligoclonal restriction of CTL cultures with lytic activity compared to identical cytokine expanded T cells lacking leukemia–specific CTL activity (Figure 3). Conclusion: A polyclonal leukemia specific CTL can be generated with IL-15 alone from previously expanded and partially mature CB T cells. While the cytotoxicity is preserved compared to those primed/expanded by multiple cytokines the specificity is higher in IL-15 alone. Notably, the CTL activity is not confined to a single cellular subset and possibly the most significant activity resides in the TCRγδ expressing T cell population. Disclosures: No relevant conflicts of interest to declare.

Download Full-text

Redirected CD4+ T Cells towards HLA Class I-Restricted WT1 Epitope Successfully Display Multifactorial Helper Function for the Enhancement of Antileukemia Efficacy Mediated by Redirected T-Cell Based Immunocelltherapy.

Blood ◽

10.1182/blood.v120.21.3153.3153 ◽

2012 ◽

Vol 120 (21) ◽

pp. 3153-3153

Author(s):

Yukihiro Miyazaki ◽

Hiroshi Fujiwara ◽

Toshiki Ochi ◽

Sachiko Okamoto ◽

Hiroaki Asai ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Cd8 T Cells ◽

Cd4 T Cells ◽

Hla Class I ◽

Class I ◽

Leukemia Cells ◽

Proliferation And Differentiation ◽

Ifn Γ

Abstract Abstract 3153 Purpose: In antitumor adoptive immunotherapy, the utility of tumoricidal CD8+ T cells are mainly highlighted, while in tumor immunity, the importance of tumor-reactive CD4+ T cells is also well documented. However, because the number of well-characterized tumor-associated epitopes recognized by CD4+ T cells still remains small, application of tumor-reactive CD4+ T cells is limited. In order to circumvent this drawback, redirection of CD4+ T cells to well-characterized HLA class I-restricted CD8+ T-cell epitope seems promising. In this study, using an HLA class I-restricted and WT1-specific T-cell receptor (TCR) gene transfer, we, in detail, examined helper functions mediated by those gene-modified CD4+T cells in redirected T cell-based antileukemia adoptive immunotherapy. Methods: HLA-A*2402-restricted and WT1235–243-specific TCR α/β genes were inserted into our unique retroviral vector encoding shRNAs for endogenous TCRs (WT1-siTCR vector), and was employed for gene-modification both of CD4+ and CD8+ T cells to express WT1-specific TCR. (1) WT1 epitope-responsive cytokine production mediated by WT1-siTCR-transduced CD4+ T cells (WT1-siTCR/CD4) was measured using bead-based immunoassay and ELISA assay. (2) WT1 epitope-ligation induced co-stimulatory molecules by WT1-siTCR/CD4 was assessed using flow cytometry. (3) Impacts on WT1 epitope and leukemia-specific responses; cytocidal activity, proliferation and differentiation into memory T-cell phenotype, mediated by WT1-siTCR-transduced CD8+ T cells (WT1-siTCR/CD8) provided by concurrent WT1-siTCR/CD4 were assessed using 51Cr-release assay, CD107a/intracellular IFN-γ assay, CFSE dilution assay and flow cytometry. (4) WT1 epitope-ligation triggered chemokine production mediated by WT1-siTCR/CD4 was assessed using real-time PCR, then chemotaxis mediated by WT1-siTCR/CD8 in response to those chemokines was assessed using a transwell experiment. (5) In vivo tumor trafficking mediated by WT1-siTCR/CD4 was assessed using bioluminescence imaging assay. (6) Finally, WT1-siTCR/CD4-caused in vivo augmentation of antileukemia functionality mediated by WT1-siTCR/CD8 was assessed similarly using a xenografted mouse model. Results: WT1-siTCR/CD4 showed a terminal effector phenotype; positive for transcription factor T-bet, but negative for Bcl-6 or Foxp3. Upon recognition of WT1 epitope, WT1-siTCR/CD4 produced Th1, but not Th2 cytokines in the context of HLA-A*2402, which simultaneously required HLA class II molecules on target cells. WT1 epitope-ligation enhanced WT1-siTCR/CD4 to express cell-surface OX40. In the presence of WT1-siTCR/CD4, but not non-gene-modified CD4, effector functions mediated by WT1-siTCR/CD8 in response to WT1 epitope and leukemia cells, including cytocidal activity based on CD107a expression and IFN-γ production was enhanced. Such augmentation was mediated by humoral factors produced by WT1 epitope-ligated WT1-siTCR/CD4. Additionally, proliferation and differentiation into memory phenotype, notably CD45RA- CD62L+ central memory phenotype, mediated by WT1-siTCR/CD8 in response to both WT1 epitope and leukemia cells were also augmented, accompanied with increased expression of intracellular Bcl-2 and cell-surface IL-7R. Next, CCL3/4 produced by activated WT1-siTCR/CD4 triggered chemotaxis of WT1-siTCR/CD8 which express the corresponding receptor, CCR5. Using bioluminescence imaging, intravenously infused WT1-siTCR/CD4 successfully migrated towards leukemia cells inoculated in a NOG mouse. Finally, co-infused WT1-siTCR/CD4 successfully augmented immediate accumulation towards leukemia cells and antileukemia reactivity mediated by WT1-siTCR/CD8 in a xenografted mouse model. Conclusion: Using GMP grade WT1-siTCR vector, redirected CD4+ T cells to HLA class I-restricted WT1 epitope successfully recognized leukemia cells and augmented in vivo antileukemia functionality mediated by similarly redirected CD8+ T cells, encompassing tumor trafficking, cytocidal activity, proliferation and differentiation into memory cells. The latter seem to support the longevity of transferred antileukemia efficacy. Taking together, coinfusion of redirected CD4+ T cells to HLA class I-restricted WT1 epitope seems feasible and advantageous for the successful WT1-targeting redirected T cell-based immunotherapy against human leukemia. Disclosures: No relevant conflicts of interest to declare.

Download Full-text

Immune surveillance of the liver by T cells

Science Immunology ◽

10.1126/sciimmunol.aba2351 ◽

2020 ◽

Vol 5 (51) ◽

pp. eaba2351

Author(s):

Xenia Ficht ◽

Matteo Iannacone

Keyword(s):

T Cells ◽

T Cell ◽

Cross Talk ◽

Immune Cell ◽

Current Knowledge ◽

Immune Surveillance ◽

Neoplastic Diseases ◽

Cell Responses ◽

Immune Cell Subsets ◽

Invariant T Cells

The liver is the target of several infectious, inflammatory, and neoplastic diseases, which affect hundreds of millions of people worldwide and cause an estimated death toll of more than 2 million people each year. Dysregulation of T cell responses has been implicated in the pathogenesis of these diseases; hence, it is critically important to understand the function and fate of T cells in the liver. Here, we provide an overview of the current knowledge on liver immune surveillance by conventional and invariant T cells and explore the complex cross-talk between immune cell subsets that determines the balance between hepatic immunity and tolerance.

Download Full-text

Osteoclast-Induced Immunosuppression Occurs through Dysregulation of Immune Checkpoint Axes in Multiple Myeloma

Blood ◽

10.1182/blood-2019-130462 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 4382-4382

Author(s):

Kenta Mukaihara ◽

Mattia D'Agostino ◽

Cristina Panaroni ◽

Keertik Fulzele ◽

Tomoaki Mori ◽

...

Keyword(s):

Multiple Myeloma ◽

T Cells ◽

T Cell ◽

Immune Cells ◽

Immune Checkpoint ◽

Th17 Cells ◽

Immune Surveillance ◽

Color Flow ◽

Osteolytic Bone Disease ◽

Ifn Γ

Osteolytic bone disease has a significant impact on both quality of life and overall survival of patients with multiple myeloma (MM). Given that bone and immune cells share the same microenvironment and interact with each other via cytokines, signaling molecules and regulatory proteins in the bone marrow (Tsukasaki M et al., Nat Rev Immunol 2019), we sought to elucidate the effect of osteoclasts (OCs) on the immune microenvironment. Specifically, T cells significantly suppress differentiation of OCs through signaling crosstalk between RANKL and IFN-γ (Takayanagi H et al., Nature 2000). However, little is known regarding the effect of OCs on the immune system. Here we investigated the effect of OCs on immune cells, especially on T cells. Dysregulation of several checkpoint molecules has been shown in MM (Kwon M et al., J Immunol 2017). We, therefore, assessed the expression of various immune checkpoint receptors, such as PD-1, TIGIT, OX40 and CD137 on T cells in co-culture with or without autologous OCs by multi-color flow cytometry. OC co-culture significantly increased co-inhibitory checkpoint (PD-1 and TIGIT) through direct contact, while decreasing co-stimulatory checkpoint (OX40 and CD137) in CD3+ T cells. Expression of the checkpoint ligand, PD-L1 was significantly increased on MM cells in the presence of both T cells and OCs compared to the presence of T cells alone. Conversely, OCs in the absence of T cells did not induce significant increase of PD-L1 on MM cells. According to previous literature, PD-L1 expression on MM cells is induced in part by IFN-γ (Liu J et al., Blood 2007). Interestingly, our data demonstrated that OCs activated IFN-γ producing T cells in co-culture conditions. Furthermore, we observed that T cell and OC-mediated upregulation of PD-L1 on MM cells was partially reversed by using IFN-γ neutralizing antibody. This implies that OCs indirectly induce PD-L1 upregulation on MM cells by enhancing IFN-γ secretion from T cells. In addition, we found that naïve CD4+ T cells have a higher propensity to differentiate into Th17 lineage in the presence of Th17 differentiation cytokines when co-cultured with OCs. Moreover, we observed an increased expansion of Th17 cells in co-culture with OCs. Those Th17 cells also showed a similar pattern of dysregulation of immune checkpoint axes. Our study demonstrates that OCs positively regulate co-inhibitory checkpoint molecules and negatively regulate co-stimulatory molecules on T cells. These findings indicate that OCs play an important role in inhibiting T cell-mediated antitumor immunity. Targeting OCs may help restore impaired immune surveillance in MM in addition to their critical role in preventing lytic bone lesions in MM. These data may support the role of antiresorptives in immune surveillance with indirect anti-tumor effects. Disclosures No relevant conflicts of interest to declare.

Download Full-text

CD8+ T-cell-mediated immunoediting influences genomic evolution and immune evasion in murine gliomas

10.1101/705152 ◽

2019 ◽

Author(s):

J. Robert Kane ◽

Junfei Zhao ◽

Takashi Tsujiuchi ◽

Brice Laffleur ◽

Aayushi Mahajan ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Tumor Cell ◽

Immune Evasion ◽

Cd8 T Cells ◽

Immune Cell ◽

Cd8 T Cell ◽

Mapk Signaling ◽

Immune Recognition ◽

Cancer Immunoediting

AbstractCancer immunoediting shapes tumor progression by the selection of tumor cell variants that can evade immune recognition. Given the immune evasion and intra-tumor heterogeneity intrinsic to gliomas, we hypothesized that CD8+ T-cells mediate immunoediting in these tumors. We evaluated glioma progression in the absence of CD8+ T-cells by depleting this immune cell population in transgenic murine gliomas. Upon transplantation, gliomas that developed in the absence of CD8+ T-cells engrafted poorly in recipients with intact immunity but engrafted well in those with CD8+ T-cell depletion. Gliomas developed in absence of CD8+ T-cells exhibited increased chromosomal instability, MAPK signaling, gene fusions, and macrophage/microglial infiltration. MAPK activation correlated with macrophage/microglial recruitment in this model and in the human disease. Our results indicate that CD8+ T-cells mediate immunoediting during gliomagenesis, influencing the genomic stability of glioma and its microenvironment, leading to immune evasion.SignificanceImmune evasion renders cancer resistant to anti-tumoral immunity. Therapeutic intervention often fails for gliomas because of the plasticity of tumor cell variants that resist immune surveillance. Our results demonstrate a mechanism of immune evasion in gliomas that derives from CD8+ T-cells during the development and progression of this disease.

Download Full-text