A Human In Vitro T Cell Exhaustion Model for Assessing Immuno-Oncology Therapies

2020 ◽  
pp. 89-101 ◽  
Author(s):  
Lynne S. Dunsford ◽  
Rosie H. Thoirs ◽  
Emma Rathbone ◽  
Agapitos Patakas
Keyword(s):  
T Cell ◽  
T Cell Exhaustion ◽  
Cell Exhaustion

scholarly journals 735 Identification of a small molecule that prevents T cell exhaustion using machine learning algorithms paired with high-resolution single cell RNAseq

2021 ◽  
Vol 9 (Suppl 3) ◽  
pp. A766-A766
Author(s):  
Isabelle Le Mercier ◽  
Sunny Sun ◽  
Dongmei Xiao ◽  
Laura Isacco ◽  
Daniel Treacy ◽  
...  
Keyword(s):  
T Cell ◽  
Low Dose ◽  
Machine Learning Algorithms ◽  
High Dose ◽  
T Cell Exhaustion ◽  
Tcr Signaling ◽  
Compound A ◽  
Cell Exhaustion

BackgroundT cell responses are tightly regulated and require a constant balance of signals during the different stages of their activation, expansion, and differentiation. As a result of chronic antigen exposure, T cells become exhausted in solid tumors, preventing them from controlling tumor growth.MethodsWe identified a transcriptional signature associated with T cell exhaustion in patients with melanoma and used our proprietary machine learning algorithms to predict molecules that would prevent T cell exhaustion and improve T cell function. Among the predictions, an orally available small molecule, Compound A, was highly predicted.ResultsCompound A was tested in an in vitro T cell Exhaustion assay and shown to prevent loss of proliferation and expression of immune checkpoint receptors. Transcriptionally, Compound A-treated cells looked indistinguishable from conventionally expanded, non-exhausted T cells. However, when assessed in a classical T cell activation assay, Compound A demonstrated dose dependent activity. At low dose, Compound A was immuno-stimulatory, allowing cells to divide further by preventing activation induced cell death. At higher doses, Compound A demonstrated immuno-suppressive activity preventing early CD69 upregulation and T cell proliferation. All together, these observations suggest that Compound A prevented exhaustion with a mechanism of action involving TCR signaling inhibition. While cessation of TCR signaling or rest has been recently associated with improved CAR-T efficacy by preventing or reversing exhaustion during the in vitro manufacturing phase, it is unclear if that mechanism would translate in vivo.Compound A was evaluated in the CT26 and MC38 syngeneic mouse models alongside anti-PD1. At low dose Compound A closely recapitulated anti-PD1 mediated cell behavior changes by scRNA-seq and flow cytometry in CT26 mice. At high dose, Compound A led to the accumulation of naive cells in the tumor microenvironment (TME) confirming the proposed mechanism of action. Low dose treatment was ineffective in MC38 mouse model but a pulsed treatment at high dose also recapitulated anti-PD1 activity in most animals. Importantly, we identified a new T cell population responding to anti-PD1 that was particularly increased in the MC38 mouse model; Compound A treatment also impacted this population.ConclusionsThese data confirm that mild TCR inhibition either suboptimal or fractionated can prevent exhaustion in vivo. However, this approach has a very limited window of activity between immuno-modulatory and immuno-suppressive effects, thereby limiting potential clinical benefit. Finally, these results demonstrate that our approach and platform was able to predict molecules that would prevent T cell exhaustion in vivo.

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.

scholarly journals Repeated stimulation or tonic-signaling chimeric antigen receptors drive regulatory T cell exhaustion

2020 ◽  
Author(s):  
Caroline Lamarche ◽  
German E. Novakovsky ◽  
Christopher N. Qi ◽  
Evan W. Weber ◽  
Crystal L. Mackall ◽  
...  
Keyword(s):  
T Cell ◽  
Regulatory T Cell ◽  
T Cell Exhaustion ◽  
Antigen Receptors ◽  
Chronic Stimulation ◽  
T Cell Therapy ◽  
Cell Exhaustion ◽  
Graft Versus Host

AbstractRegulatory T cell (Treg) therapy is a promising approach to improve outcomes in transplantation and autoimmunity. In conventional T cell therapy, chronic stimulation can result in poor in vivo function, a phenomenon termed exhaustion. Whether or not Tregs are also susceptible to exhaustion, and if so, if this would limit their therapeutic effect, was unknown. We studied how two methods which induce conventional T cell exhaustion – repetitive stimulation or expression of a tonic-signaling chimeric antigen receptor (CAR) – affect human Tregs. With each repetitive polyclonal stimulation Tregs progressively acquired an exhausted phenotype, and became less suppressive in vitro. Tregs expressing a tonic-signaling CAR rapidly acquired an exhausted phenotype and had major changes in their transcriptome and metabolism. Although tonic-signaling CAR-Tregs remained stable and suppressive in vitro, they lost in vivo function, as tested in a model of xenogeneic graft-versus-host disease. The finding that human Tregs are susceptible to exhaustion has important implications for the design of Treg adoptive immunotherapy strategies.

scholarly journals Allicin May Promote Reversal of T-Cell Dysfunction in Periodontitis via the PD-1 Pathway

2021 ◽  
Vol 22 (17) ◽  
pp. 9162
Author(s):  
Shankargouda Patil ◽  
Mohammed E. Sayed ◽  
Maryam H. Mugri ◽  
Khalaf F. Alsharif ◽  
Arif Salman ◽  
...  
Keyword(s):  
T Cell ◽  
In Silico ◽  
Gingival Crevicular Fluid ◽  
T Cell Exhaustion ◽  
Inhibitory Interaction ◽  
Cell Exhaustion ◽  
In Silico Docking ◽  
T Cell Dysfunction ◽  
Crevicular Fluid

We evaluated the role of allicin in periodontitis using an in silico and in vitro design. An in silico docking analysis was performed to assess the plausible interactions between allicin and PD-L1. The cytokine profile of gingival crevicular fluid (GCF) samples obtained from periodontitis patients was estimated by cytometric bead array. CD3+ lymphocytes isolated from the peripheral blood were sorted and characterized using immunomagnetic techniques. Cultured and expanded lymphocytes were treated with the GCF samples to induce T-cell exhaustion. Optimum concentrations of allicin were added to exhausted lymphocytes to compare the expression of TIM-3 and LAG-3 gene expression at baseline and post-treatment. Allicin was found to bind to the PD-L1 molecule as revealed by the in-silico experiment, which is possibly an inhibitory interaction although not proven. GCF from periodontitis patients had significantly higher concentrations of TNF-α, CCL2, IL-6, IFN-γ, and CXCL8 than controls. GCF treatment of CD3+ lymphocytes from the periodontitis patients significantly increased expression of T-cell exhaustion markers TIM-3 and LAG-3. Allicin administration with GCF treatment resulted in significant lowering of the expression of exhaustion markers. Allicin may exert an immunostimulatory role and reverse immune-destructive mechanisms such as T-cell exhaustion.

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.

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

2021 ◽  
Author(s):  
Mirko Corselli ◽  
Suraj Saksena ◽  
Margaret Nakamoto ◽  
Woodrow E. Lomas ◽  
Ian Taylor ◽  
...  
Keyword(s):  
T Cell ◽  
Single Cell ◽  
T Cell Exhaustion ◽  
Cell Exhaustion

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 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 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.

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