scholarly journals 885 Targeting FPR2 as a novel approach for immunotherapy in pancreatic cancer female patients - studies of sexual immune dimorphism in the tumor microenvironment

2021 ◽  
Vol 9 (Suppl 3) ◽  
pp. A927-A927
Author(s):  
Dhifaf Sarhan ◽  
Fei He ◽  
Ahmed Calandigary ◽  
Enana Malki ◽  
Carlos Fernández Moro ◽  
...  
Keyword(s):  
Pancreatic Cancer ◽  
T Cells ◽  
T Cell ◽  
Sexual Dimorphism ◽  
Myeloid Cells ◽  
T Cell Exhaustion ◽  
Gi Tract ◽  
Cell Exhaustion ◽  
Tumor Phenotype

BackgroundImmunotherapy for pancreatic cancer (PC) is inefficient due to a highly immune-suppressive tumor microenvironment (TME) orchestrated by myeloid suppressor cells, which limit the infiltration and function of cytotoxic immune cells. We have evidence that accumulation of a subpopulation of myeloid cells in human pancreatic lesions is associated with immune-exclusive tumor phenotype and effector T cell exhaustion by mechanisms involving the G-coupled protein receptor formyl peptide receptor 2 (FPR2), exclusively in women. We hypothesize that female FPR2+ myeloid cells in tumors induce immune exhaustion and contribute to immune-cold tumor phenotype.MethodsTo test our hypothesis, we first investigated the FPR2 RNA and protein expression in PC transcriptomic data and in murine and human PC tissues. Further, in vitro cytokine differentiated, alternatively tumor conditioned myeloid cells (TCM) were co-cultured with T cells to mimic their interaction in the TME. In vivo, PC cells were injected subcutaneously in FPR2 WT and KO mice to study tumor progression and the immune landscape in male vs. female mice. Later, human myeloid cells were treated with FPR2 agonists and antagonists to study the interaction mechanisms in detail.ResultsWe found high FPR2 expression in tumor compared to healthy tissues and higher in women compared to men. In mice and human, FPR2+ myeloid cells were associated with immune cold-exclusive and cold-ignored tumor phenotype in women and men, respectively. Notably, analysis in PC and other gastrointestinal (GI)-tract cancers revealed a significant association of FPR2 expression and poor survival only in women, emerging the potential impact of sex factors in the TME. Such sexual dimorphism in the TME was associated with T cell exhaustion apparent by high expression of TIM3 and PD1. In vitro, FPR2-agonist treated myeloid-suppressive cells induced TIM3 and PD1 expression in T cells specifically in female T cells. However, a significant repression of TIM3 and a trend of PD1 expression was observed in T cells when interacting with FPR2-inhibited or -deficient myeloid cells. Finally, tumor progression was significantly slower in FPR2 KO female mice compared to WT and male FPR2 WT and KO mice.ConclusionsIn this study, we have shown that sex differences are involved in shaping the TME in PC, where sexual dimorphism is still a largely unknown area allowing novel personalized/sex-specific immunotherapies. We found that FPR2 is highly involved in T cell exhaustion and can potentially be a therapeutic target for immunotherapy in women developing PC and other GI-tract cancers.Ethics ApprovalThe study was approved by the regional ethics review board in Stockholm (Dnr2020-06587 and Dnr2013.977-31.1) and the Swedish Board of Agriculture and regional ethical committee (10681-2020).

scholarly journals Single cell multiomic analysis of T cell exhaustion in vitro

2019 ◽  
Author(s):  
Mirko Corselli ◽  
Suraj Saksena ◽  
Margaret Nakamoto ◽  
Woodrow E. Lomas ◽  
Ian Taylor ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Single Cell ◽  
T Cell Activation ◽  
T Cell Exhaustion ◽  
Chronic Stimulation ◽  
Cell Exhaustion ◽  
Car T ◽  
The Impact

AbstractA key step in the clinical production of CAR-T cells is the expansion of engineered T cells. To generate enough cells for a therapeutic product, cells must be chronically stimulated, which raises the risk of inducing T-cell exhaustion and reducing therapeutic efficacy. As protocols for T-cell expansion are being developed to optimize CAR T cell yield, function and persistence, fundamental questions about the impact of in vitro manipulation on T-cell identity are important to answer. Namely: 1) what types of cells are generated during chronic stimulation? 2) how many unique cell states can be defined during chronic stimulation? We sought to answer these fundamental questions by performing single-cell multiomic analysis to simultaneously measure expression of 39 proteins and 399 genes in human T cells expanded in vitro. This approach allowed us to study – with unprecedented depth - how T cells change over the course of chronic stimulation. Comprehensive immunophenotypic and transcriptomic analysis at day 0 enabled a refined characterization of T-cell maturational states (from naïve to TEMRA cells) and the identification of a donor-specific subset of terminally differentiated T-cells that would have been otherwise overlooked using canonical cell classification schema. As expected, T-cell activation induced downregulation of naïve-associated markers and upregulation of effector molecules, proliferation regulators, co-inhibitory and co-stimulatory receptors. Our deep kinetic analysis further revealed clusters of proteins and genes identifying unique states of activation defined by markers temporarily expressed upon 3 days of stimulation (PD-1, CD69, LTA), markers constitutively expressed throughout chronic activation (CD25, GITR, LGALS1), and markers uniquely up-regulated upon 14 days of stimulation (CD39, ENTPD1, TNFDF10). Notably, different ratios of cells expressing activation or exhaustion markers were measured at each time point. These data indicate high heterogeneity and plasticity of chronically stimulated T cells in response to different kinetics of activation. In this study, we demonstrate the power of a single-cell multiomic approach to comprehensively characterize T cells and to precisely monitor changes in differentiation, activation and exhaustion signatures in response to different activation protocols.

Pseudo-Exhaustion Of CD8+ T Cells in AML

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 2615-2615 ◽  
Author(s):  
Felix S. Lichtenegger ◽  
Frauke M. Schnorfeil ◽  
Katharina Emmerig ◽  
Julia S. Neitz ◽  
Barbara Beck ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cd8 T Cells ◽  
Peptide Pool ◽  
Primary Diagnosis ◽  
Refractory Disease ◽  
T Cell Exhaustion ◽  
Cell Exhaustion ◽  
Inhibitory Molecules

Abstract The prognosis of acute myeloid leukemia (AML), particularly when associated with adverse chromosomal or molecular aberrations, is poor due to a high relapse rate after induction chemotherapy. Postremission therapy for elimination of minimal residual disease remains a major challenge. Immunotherapeutic strategies such as dendritic cell (DC) vaccination aim at the stimulation of AML-specific immunity, especially of CD8+ T cells. However, the functionality of these cells in AML patients is not well described. Recently, T cell exhaustion has been suggested to contribute to immune evasion in various solid and hematological malignancies. Primarily demonstrated in chronic viral infections, exhausted T cells are characterized by an increased expression of several inhibitory molecules, reduced proliferation and an impaired capability of cytokine secretion and cytotoxicity. In order to characterize T cell exhaustion in AML at primary diagnosis and during refractory disease, we assessed the phenotype and effector function of CD8+ T cells by flow cytometry. Surface expression of the inhibitory molecules CD244 (2B4), CD160, PD-1, TIM-3 and LAG-3 was determined. T cell proliferation and production of the cytokines IFN-γ, TNF-α and IL-2 were measured in response to different stimuli. Results were compared to healthy controls (HCs), while untreated HIV-infected patients served as positive controls for an exhausted state of CD8+ T cells. To specify the effect of DCs on the state of T cell exhaustion in AML, we cocultured in vitro generated DCs with autologous T cells from primary diagnosis for four days. Compared to HCs, we detected similarly increased frequencies of CD244- and TIM-3-positive CD8+ T cells in AML and HIV patients (CD244: HCs 37±17%, AML primary diagnosis/refractory disease 72±21%/67±9%, HIV 70±7%; TIM-3: HCs 1.1±1.5%, AML primary diagnosis/refractory disease 2.9±2.2%/4.7±4.4%, HIV 2.9±2.4%; mean±SD). In refractory AML, we additionally observed an increased frequency of CD8+ T cells positive for CD160 and PD-1 (CD160: HCs 19±9%, AML refractory disease 32±8%; PD-1: HCs 21±8%, AML refractory disease 50±25%). In our functional analyses, however, T cells from AML patients and HCs were equally able to produce IFN-γ, TNF-α and IL-2 upon in vitro stimulation with a CEFT peptide pool, PMA/Ionomycin or anti-CD3/CD28. Using the CEFT peptide pool for stimulation, we even measured an increase in proliferation of T cells from AML patients compared to T cells from HCs and HIV-infected patients. The in vitro stimulation of AML cells with DCs generated from autologous monocytes resulted in a further upregulation of the molecules PD-1 and TIM-3. In summary, we found an increased overall expression of inhibitory surface molecules associated with T cell exhaustion on CD8+ T cells of AML patients at primary diagnosis and with refractory disease, which was further enhanced by in vitro stimulation with DCs. In contrast, no impairment of functionality was detected, as determined by proliferation and cytokine secretion assays. We therefore hypothesize that bulk CD8+ T cells in AML are in a status of activation, not exhaustion. Disclosures: No relevant conflicts of interest to declare.

514 Disturbed Mitochondrial Dynamics Rewire the Epigenetic Program for CD8+ TIL Exhaustion

2020 ◽  
Vol 8 (Suppl 3) ◽  
pp. A550-A550
Author(s):  
Yi-Ru Yu ◽  
Haiping Wang ◽  
Fabien Franco ◽  
Ping-Chih Ho
Keyword(s):  
T Cells ◽  
T Cell ◽  
De Novo ◽  
Mitochondrial Dynamics ◽  
Glucose Deprivation ◽  
Cell Mediated Immunity ◽  
T Cell Exhaustion ◽  
Cell Exhaustion ◽  
Cell Immunity

BackgroundCancer immunotherapy, including checkpoint blockade and adoptive transfer of tumor-reactive T cells, represents a paradigm shift in the treatment of malignancies in recent years, and yields remarkable responses by reawakening anti-tumor immunity in established tumors. Nevertheless, a significant portion of patients are refractory to cancer immunotherapies, which may be in part due to the persistent impairment of anti-tumor effector functions in T cells, a phenomenon referred to as T cell exhaustion. Emerging evidence reveal that alterations in global chromatin accessibility and de novo DNA methylation patterns are keys events to drive development of T cell exhaustion under chronic antigenic stresses. However, it remains elusive how T cells engage epigenetic reprogramming to orchestrate exhausted state.MethodsHere, we examined the mitochondrial fitness in CD8+ TILs with mitoTrackers.ResultsWe found that tumor-infiltrating tumor-reactive T cells with accumulation of damaged mitochondria, characterized by increased mitochondrial mass but reduced mitochondrial membrane potential and cristae, display more severe exhausted phenotypes, including decreased proliferation capacity, reduced cytokine production and up-regulation of co-inhibitory receptors. The accumulation of damaged mitochondria is in part due to the deficiency of mitophagy machinery. Importantly, we found that the accumulation of dysfunctional mitochondria is corelated to the specificity and affinity of antigen, and also supported by the PD-1 expression. Moreover, the combination of glucose deprivation, hypoxia and TCR signaling in vitro can drastically weaken T cell immunity with the accumulation of dysfunctional mitochondria as seen in TILs previously. Furthermore, T cells with accumulation of damaged mitochondria, generated artificially by Oligomycin A and Mdivi-1, also exhibit persistent exhaustion features. Ultimately, supplementation with nicotinamide riboside enhances T cell mitochondrial fitness and improved responsiveness to anti-PD-1 treatment.ConclusionsTaken together, our study suggests that mitochondrial fitness is pivotal for T cell-mediated immunity and the accumulation of dysfunctional mitochondria could result in exhaustion phenotypes in T cells. And our findings also provide pillars for better harnessing T cell immune responses with metabolic regulations for immunotherapy.

100 Drug-regulatable engineered T cells eliminate CD33+ and CD33ΔE2+ AML

2020 ◽  
Vol 8 (Suppl 3) ◽  
pp. A111-A111
Author(s):  
Jacob Appelbaum ◽  
Wai-Hang Leung ◽  
Unja Martin ◽  
Kaori Oda ◽  
Giacomo Tampella ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Lines ◽  
Splice Variants ◽  
Antigen Binding ◽  
T Cell Exhaustion ◽  
Cell Exhaustion ◽  
Car T

BackgroundBioengineered T cell treatments for acute myeloid leukemia (AML) are challenged by near universal expression of leukemia antigens on normal hematopoietic stem/progenitor cells:1 2 ‘on target/off tumor‘ activity may cause myelosuppression while sustained antigen exposure can lead to T cell exhaustion.3 In addition, splicing variants may allow antigen escape. We hypothesize that by using a novel CD33-C2-specific single domain VHH antibody as the antigen targeting domain in dimerizing agent-regulated immunoreceptor complex T cells (DARIC T cells), we will enable pharmacologically-controllable targeting of CD33, allowing eradication of leukemia expressing either of the major splice variants of CD33: i.e., full-length CD33 or CD33ΔE2.MethodsWe engineered DARIC-expressing lentiviral vectors containing encoding separated CD33-C2-specific antigen binding and 41BB-CD3zeta signaling chains that heterodimerize following addition of rapamycin via embedded FKBP12 and FRB* domains.4 Peripheral blood mononuclear cells were stimulated with IL-2, anti-CD3, and anti-CD28 antibodies 24h prior to transduction with DARIC33 lentiviral vector. Surface expression of antigen binding or signaling chains was assessed using biotinylated CD33, or antibodies to VHH-domains or FRB* respectively. Rapamycin-dependent in vitro activity was measured by IFNg release. To evaluate in vivo activity, NSG mice injected with 1 × 105 MOLM-14/luc cells were treated 5-7 days later with 1 × 107 DARIC33 T cells in the presence or absence of rapamycin and tumor progression followed by luciferase activity.ResultsDARIC33+ T cells bound biotinylated-CD33, anti-VHH and anti-FRB* antibodies. Rapamycin addition increased expression of both signaling and antigen-recognition chains, suggesting augmented receptor stability in the presence of dimerizing drug. In the presence of rapamycin, coculture of DARIC33 T cells with cell lines expressing either full length or CD33ΔE25 showed equivalent rapamycin-dependent activation, demonstrating DARIC33 responds to both splice variants. Titration experiments showed rapamycin-dependent activation with EC50 = 25pM. Negligible IFNg release was observed in the absence of drug. DARIC33 T cells significantly extended survival of AML-bearing mice, but only when treated with rapamycin. The DARIC33 T cells were activated in vivo by sub-immunosuppressive rapamycin dosing, as weekly or 0.1 mg/kg QOD dosing led to similar levels of tumor suppression.ConclusionsDARIC33 T cells appear to be potent antileukemic agents: they are activated by AML cell lines in vitro as demonstrated by cytokine release and cytotoxicity, and significantly extend survival in an aggressive xenograft model. Temporal control provided by the DARIC architecture promises to enhance safety and potentially efficacy of CAR T therapy for AML, for example by enabling hematopoietic recovery or providing T cell rest.ReferencesPerna F, Berman SH, Soni RK, Mansilla-Soto J, Eyquem J, Hamieh M, et al. Integrating proteomics and transcriptomics for systematic combinatorial chimeric antigen receptor therapy of AML. Cancer Cell 2017 Oct 9;32(4):506–519.e5.Haubner S, Perna F, Köhnke T, Schmidt C, Berman S, Augsberger C, et al. Coexpression profile of leukemic stem cell markers for combinatorial targeted therapy in AML. Leukemia. 2019 Jan;33(1):64.Lamarche C, Novakovsky GE, Qi CN, Weber EW, Mackall CL, Levings MK. Repeated stimulation or tonic-signaling chimeric antigen receptors drive regulatory T cell exhaustion. bioRxiv. 2020 Jun 28;2020.06.27.175158.Leung W-H, Gay J, Martin U, Garrett TE, Horton HM, Certo MT, et al. Sensitive and adaptable pharmacological control of CAR T cells through extracellular receptor dimerization. JCI Insight [Internet]. 2019 Jun 6 [cited 2019 Jun 11];4(11). Available from: https://insight.jci.org/articles/view/124430Pérez-Oliva AB, Martínez-Esparza M, Vicente-Fernández JJ, Corral-San Miguel R, García-Peñarrubia P, Hernández-Caselles T. Epitope mapping, expression and post-translational modifications of two isoforms of CD33 (CD33M and CD33m) on lymphoid and myeloid human cells. Glycobiology 2011;21(6):757–770.

scholarly journals 251 The use of small molecule inhibitors to target novel pathways in exhausted T cells for immuno-oncology therapeutic intervention

2021 ◽  
Vol 9 (Suppl 3) ◽  
pp. A271-A271
Author(s):  
Francis Acklam ◽  
Joanne Hay ◽  
Darryl Turner ◽  
Mark Barbour ◽  
Preeti Singh ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Small Molecule ◽  
Therapeutic Intervention ◽  
Checkpoint Inhibitor ◽  
Great Promise ◽  
Cell Phenotype ◽  
T Cell Exhaustion ◽  
Cell Exhaustion

BackgroundThe ability to reverse exhaustion in CD8+ T cells holds great promise for therapeutic intervention in oncology. Indeed, treatment with therapeutics targeted at checkpoint inhibitors, such as Nivolumab (anti-PD-1), have shown great promise in the treatment of a subset of individuals and tumour types. However, pre-clinical success does not always translate to success in clinical trials and resistance to these approaches is prevalent. As such, there is a pressing need to develop novel approaches that target alternative pathways for use alone or potentially in combination with checkpoint inhibitor modulation. A secondary need is the requirement for advanced assays that accurately recapitulate the pathways and cell phenotypes prevalent in the tumour environment.MethodsHere we describe the characteristics of an in vitro human T cell exhaustion assay whereby in vitro stimulated T cells phenotypically and functionally recapitulate the exhausted T cells found within the tumour microenvironment. We also demonstrate the effect of checkpoint blockade as well as small molecule inhibition of a novel target on the exhausted T cell phenotype.ResultsIn this assay, exhaustion can be partially but not fully reversed by treatment with anti-PD-1 alone. In addition, we demonstrate the effect of a small molecule inhibitor targeting IKZF3, a transcription factor shown to be upregulated in T cell exhaustion, on reversing T cell exhaustion alone and in combination with checkpoint inhibitor blockade.ConclusionsThese assays and approaches enable investigation into the ability of compounds to influence reversal of T cell exhaustion where anti-PD-1 treatment does not fully reverse the exhausted phenotype and offers the ability to test combination therapy approaches.AcknowledgementsThis work was aided by the valuable input and insight of Professor Stephen Anderton.Ethics ApprovalThe study obtained ethics approval from West Midlands – Black Country Research Ethics Committee under IRAS project ID 270936. All donors gave informed consent before taking part.

scholarly journals Treatment-Free Intervals Mitigate T-Cell Exhaustion Induced By Continuous CD19xCD3-BiTE® Construct Stimulation in Vitro

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 44-45
Author(s):  
Nora Zieger ◽  
Alyssa Nicholls ◽  
Jan Wulf ◽  
Gerulf Hänel ◽  
Maryam Kazerani Pasikhani ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Research Funding ◽  
Cell Function ◽  
Granzyme B ◽  
T Cell Exhaustion ◽  
Specific Lysis ◽  
Cell Exhaustion ◽  
T Cell Function

The bispecific T-cell engager (BiTE®) blinatumomab is approved for treatment of relapsed/refractory B-cell precursor acute lymphoblastic leukemia and applied as continuous infusion over 28 days. The overall response rate to blinatumomab reported in clinical trials was 43 % and correlated to T-cell expansion (Zugmaier et al. 2015). In chronic viral infections, continuous antigen stimulation induces T-cell exhaustion, defined by phenotypic changes and functional impairment (Wherry 2011). Thus, we hypothesized that continuous BiTE® construct stimulation leads to T-cell exhaustion and that a treatment-free interval (TFI) reverses progressive T-cell dysfunction. To simulate continuous application of a BiTE® construct in vitro, T-cell long-term co-cultures were set up. Healthy donor T cells were stimulated in the presence of CD19+ OCI-Ly1 cells for 28 days with AMG 562, a half-life extended CD19 and CD3 specific BiTE® construct. T cells were harvested from the co-culture every 3-4 days between day 7 and 28 and assessed for markers of T-cell exhaustion: (1) AMG 562-mediated cytotoxicity of T cells was evaluated as specific lysis of CD19+ Ba/F3 target cells after 3 days, (2) T-cell expansion during the cytotoxicity assay was calculated as fold change (FC) of CD2+ counts, (3) Cytokine secretion of AMG 562-stimulated T cells was evaluated in co-culture supernatants by cytometric bead array (CBA) or after PMA/Ionomycine stimulation via intracellular cytokine staining (ICCS), (4) T-cell metabolic fitness was determined by Mito- and Glycolytic Stress Test using a Seahorse Analyzer, and (5) expression of the exhaustion-related transcription factor TOX was assessed by multiparameter flow cytometry. In order to assess the effect of a TFI on T-cell function, we cultured T cells and CD19+ OCI-Ly1 cells in the absence of AMG 562 from day 7-14 and 21-28 and compared their activity to T cells stimulated continuously with AMG 562. On day 7 of continuous (CONT) AMG 562 stimulation, we observed high cytotoxic and proliferative potential (% specific lysis=93±0.2, FC=2.9±0.2) as well as high IFN-g and TNF-a secretion analyzed by ICCS (% CD8+IFN-g+TNF-a+=23±6.7). However, cytotoxicity and proliferation decreased gradually until day 28 (% specific lysis=28±8.9; FC=0.6±0.1). CBA analysis confirmed decreasing secretion of IFN-g (day 3: 61113±12482, day 24: 3085±1351 pg/ml) and TNF-a (day 3: 1160±567, day 24: 43±7.6 pg/ml) as well as decreased IL-2 and granzyme B levels in culture supernatants. We furthermore observed highest mitochondrial fitness and basal glycolysis in T cells on day 7 of stimulation (basal OCR=2.2±0.6, maximal OCR=3.7±1.0, SRC=1.5±1.1 pmol/min/1000 cells, basal ECAR=2.0±0.4 mpH/min/1000 cells) which decreased until day 28 (basal OCR=0.4±0.2, maximal OCR=1.5±0.5, SRC=1.0±0.2 pmol/min/1000 cells, basal ECAR=0.5±0.2 mpH/min/1000 cells). In concordance, TOX increased during continuous stimulation (MFI ratio CD8+ day 7=6±0.8 to 12±0.8 on day 28). Strikingly, implementation of a TFI of 7 days led to superior cytotoxicity in T cells compared to continuously stimulated T cells (% specific lysis on day 14 CONT=34±4.2, TFI=99±2.2) and granzyme B production (CD8+; MFI ratio on day 14 CONT=124±11, TFI=303±34). Furthermore, increased proliferation during the cytotoxicity assay was observed in previously rested T cells (FC CONT=0.2±0.0, TFI=1.6±0.6). Although T cell function also decreased over time in TFI T cells, they maintained a strikingly higher cytotoxic potential (CONT=6±4.4, TFI=52±9.9) as well as higher granzyme B production (CONT=25±2, TFI=170±11) on day 28 compared to continuously stimulated T cells. In addition, TFI T cells showed increased IFN-g and TNF-a secretion after PMA/Ionomycine stimulation on day 28 (% CD8+IFN-g+TNF-a+ CONT=21±3.8, TFI=38±11.6). Our in vitro results demonstrate that continuous AMG 562 exposure negatively impacts T-cell function. Comprehensive analysis of T-cell activity in an array of functional assays suggests that continuous BiTE® construct exposure leads to T-cell exhaustion which can be mitigated through TFI. Currently, T cells from patients receiving blinatumomab are being analyzed to confirm the clinical relevance of our findings. Furthermore, RNA-Seq of continuously vs. intermittently AMG 562-exposed T cells will help us to understand underlying transcriptional mechanisms of BiTE® construct induced T-cell exhaustion. Disclosures Zieger: AMGEN Research Munich: Research Funding. Buecklein:Pfizer: Consultancy; Novartis: Research Funding; Celgene: Research Funding; Amgen: Consultancy; Gilead: Consultancy, Research Funding. Brauchle:AMGEN Inc.: Research Funding. Marcinek:AMGEN Research Munich: Research Funding. Kischel:AMGEN: Current Employment, Current equity holder in publicly-traded company, Patents & Royalties. Subklewe:Gilead Sciences: Consultancy, Honoraria, Research Funding; Pfizer: Consultancy, Honoraria; Morphosys: Research Funding; Seattle Genetics: Research Funding; AMGEN: Consultancy, Honoraria, Research Funding; Janssen: Consultancy; Roche AG: Consultancy, Research Funding; Novartis: Consultancy, Research Funding; Celgene: Consultancy, Honoraria.

scholarly journals Lineage and Spatial Mapping of Glioblastoma-associated Immunity

2020 ◽  
Author(s):  
Vidhya M. Ravi ◽  
Nicolas Neidert ◽  
Paulina Will ◽  
Kevin Joseph ◽  
Julian P. Maier ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
T Cell Activation ◽  
In Silico ◽  
Myeloid Cells ◽  
Lymphoid Cells ◽  
List Type ◽  
T Cell Exhaustion ◽  
Cellular Interactions ◽  
Cell Exhaustion

AbstractThe diversity of molecular states and cellular plasticity of immune cells within the glioblastoma environment remain poorly investigated. Here, we performed scRNA-sequencing of the immune compartment, mapping potential cellular interactions that lead to the exhausted phenotype of T cells. We identified Interleukin 10 response during T cell activation leading to the exhausted state. By use of an in-silico model, we explored cell-cell interactions and identified a subset of myeloid cells defined by high expression of HMOX1 driving T cell exhaustion. We showed a spatial correlation between T cell exhaustion and mesenchymal-like gene expression, co-located with HMOX1 expressing myeloid cells. Using human neocortical sections with myeloid cell depletion, we confirmed the functional interaction of myeloid and lymphoid cells, leading to the exhausted state of T cells. A comprehensive understanding of cellular states and plasticity of lymphoid cells in GBM aids in providing successful immunotherapeutic approaches.HighlightsLineage tracking of T cells reveal IL10 driven exhaustion in glioblastomaIn-silico modeling of spatial- and scRNA-sequencing identified a subset of HMOX1+ myeloid cells releasing IL10.T cell exhaustion is spatially enriched in mesenchymal-like tumor regions.Human neocortical sections with autograft T cell stimulation confirmed IL10 dependent T cell exhaustion in mesenchymal-like tumors.Visual Abstract

scholarly journals IMMU-34. LANDSCAPE OF GLIOBLASTOMA-ASSOCIATED IMMUNITY BY INTEGRATION OF scRNA-SEQ AND SPATIAL TRANSCRIPTOMICS

2020 ◽  
Vol 22 (Supplement_2) ◽  
pp. ii112-ii112
Author(s):  
Vidhya Ravi ◽  
Nicolas Neidert ◽  
Kevin Joseph ◽  
Juergen Beck ◽  
Oliver Schnell ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
T Cell Activation ◽  
Cell Activation ◽  
Myeloid Cells ◽  
Myeloid Cell ◽  
Lymphoid Cells ◽  
T Cell Exhaustion ◽  
Cellular Interactions ◽  
Cell Exhaustion

Abstract The diversity of molecular states and cellular plasticity of immune cells within the glioblastoma (GBM) environment remain poorly investigated. Here, we conduct deep transcriptional profiling of lymphoid and myeloid cell populations by scRNA-sequencing, map potential cellular interactions and cytokine responses that lead to the dysfunctional and exhausted phenotype of T cells. We identified Interleukin 10 (IL-10) response during T cell activation, which lead to a dysfunctional state of T cells. By the use of a novel method: The nearest functionally connected neighbor (NFCN), an in-silico model to explore cell-cell interaction, the dysfunctional/exhausted phenotype was found to be driven by subset of myeloid cells defined by high expression of HMOX1. By using spatial transcriptomic RNA-sequencing, we identified a correlation between T cell exhaustion and colocalized mesenchymal gene expression. We found that HMOX1 expressing myeloid cells occupying regions marked by T cell exhaustion. Using a human neocortical slice model with myeloid cell depletion we confirmed the functional interaction of myeloid and lymphoid cell leading to the dysfunctional state of T cells. A comprehensive understanding of cellular states and plasticity of lymphoid cells in GBM aids in providing successful immunotherapeutic approaches.

VEGF-A drives TOX-dependent T cell exhaustion in anti–PD-1–resistant microsatellite stable colorectal cancers

2019 ◽  
Vol 4 (41) ◽  
pp. eaay0555 ◽  
Author(s):  
Chang Gon Kim ◽  
Mi Jang ◽  
Youngun Kim ◽  
Galam Leem ◽  
Kyung Hwan Kim ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Ex Vivo ◽  
Immune Checkpoint Blockade ◽  
T Cell Exhaustion ◽  
Multiple Cancer ◽  
Cell Exhaustion ◽  
Cancer Settings

Although immune checkpoint blockade therapies have demonstrated clinical efficacy in cancer treatment, harnessing this strategy is largely encumbered by resistance in multiple cancer settings. Here, we show that tumor-infiltrating T cells are severely exhausted in the microsatellite stable (MSS) colorectal cancer (CRC), a representative example of PD-1 blockade–resistant tumors. In MSS CRC, we found wound healing signature to be up-regulated and that T cell exhaustion is driven by vascular endothelial growth factor-A (VEGF-A). We report that VEGF-A induces the expression of transcription factor TOX in T cells to drive exhaustion-specific transcription program in T cells. Using a combination of in vitro, ex vivo, and in vivo mouse studies, we demonstrate that combined blockade of PD-1 and VEGF-A restores the antitumor functions of T cells, resulting in better control of MSS CRC tumors.

scholarly journals 711 IGM-7354 is an anti-PD-L1 IgM antibody and IL-15 cytokine fusion that enhances NK and CD8+ T cell proliferation and tumor cytotoxicity plus potently reverses T cell exhaustion

2021 ◽  
Vol 9 (Suppl 3) ◽  
pp. A740-A740
Author(s):  
Thierry Giffon ◽  
Melanie Desbois ◽  
Dean Ng ◽  
Poonam Yakkundi ◽  
Marigold Manlusoc ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cd8 T Cells ◽  
Cynomolgus Monkey ◽  
Cd8 T Cell ◽  
High Avidity ◽  
T Cell Exhaustion ◽  
Cell Exhaustion ◽  
High Affinity

BackgroundWhile approved PD-1/PD-L1 inhibitory antibodies have demonstrated clinical efficacy in certain cancer patients, relapse following a primary response is often observed. Enhancing anti-tumor immune responses with an immunostimulatory cytokine, IL-15 is an attractive combination strategy to enhance anti-tumor NK and memory CD8+ T cell expansion and survival. We have developed IGM-7354, a high affinity, high avidity anti-PD-L1 pentameric IgM antibody with an IL-15Rα chain and IL-15 fused to the joining (J) chain, designed to deliver IL-15 to PD-L1 expressing tumors for enhancing anti-tumor immune responses.MethodsIGM-7354 was generated by grafting heavy chain variable regions of a high affinity humanized anti-PD-L1 IgG onto the IgM heavy chain framework, co-expressed with the light chain and the J chain which included a single IL-15Rα and IL-15 fusion. Binding ELISAs were performed using recombinant antigens. Human and cynomolgus monkey PBMCs were used for potency testing. Reversal of T cell exhaustion was tested using in vitro MLR. In vitro cytotoxicity assays were performed with luciferase-tagged MDA-MB-231 cells and PBMCs. In vivo pharmacodynamic studies were conducted in mice and cynomolgus monkeys.ResultsIGM-7354 bound human and cynomolgus monkey PD-L1 with the same affinity but did not bind to rat or mouse PD-L1. In addition, the IL-15 component of IGM-7354 bound to human and cynomolgus β chain of the trimeric IL-15 receptor with similar affinities, but with weaker binding affinity to rodent IL-15Rβ. Using in vitro assays with PBMCs, IGM-7354 dose dependently enhanced the proliferation of human and cynomolgus monkey NK and CD8+ T cells. Furthermore, IGM-7354 was able to reverse T cell exhaustion in an in vitro MLR beyond that of an IL-15/IL15Rα complex or anti-PD-L1 IgM or IgG alone, as demonstrated by an increase in activation and effector cytokine secretion. IGM-7354 also enhanced in vitro killing of PD-L1-expressing MDA-MB-231 breast cancer cells by human PBMCs. Pharmacodynamic studies in an MDA-MB-231 xenograft mouse model showed dose-dependent increases in circulating NK and CD8+ T cells and tumor infiltrating lymphocytes, which correlated with tumor regression. In cynomolgus monkeys, intravenous administration of IGM-7354 was well tolerated and dose dependently induced the proliferation of NK and CD8+ T cells.ConclusionsIGM-7354 stimulates NK and CD8+ T cell expansion in vitro and in vivo plus induces tumor regressions in mouse tumor models. This approach may enhance tumor localization of the immunostimulatory cytokine IL-15 through high affinity and high avidity binding to PD-L1 thereby improving anti-tumor responses and minimizing toxicity.Ethics ApprovalAll animal studies were conducted according to approved Institutional Animal Care and Use Committee (IACUC) protocols of the testing facilities.

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