Confirmation ofMycobacterium tuberculosis infection by flow cytometry after ex vivo incubation of peripheral blood T cells with an ESAT-6-derived peptide pool

BackgroundA functional cure for chronic HBV could be achieved by boosting HBV-specific immunity. In vitro studies show that immunotherapy could be an effective strategy. However, these studies include strategies to enrich HBV-specific CD8 T cells, which could alter the expression of the anti-PD-1/anti-PD-L1 antibody targets. Our aim was to determine the efficacy of PD-L1 blockade ex vivo.MethodsHBV-specific CD8 T cells were characterized ex vivo by flow cytometry for the simultaneous analysis of six immune populations and 14 activating and inhibitory receptors. Ex vivo functionality was quantified by ELISpot and by combining peptide pool stimulation, dextramers and intracellular flow cytometry staining.ResultsThe functionality of HBV-specific CD8 T cells is associated with a higher frequency of cells with low exhaustion phenotype (LAG3-TIM3-PD-1+), independently of the clinical parameters. The accumulation of HBV-specific CD8 T cells with a functionally exhausted phenotype (LAG3+TIM3+PD-1+) is associated with lack of ex vivo functionality. PD-L1 blockade enhanced the HBV-specific CD8 T cell response only in patients with lower exhaustion levels, while response to PD-L1 blockade was abrogated in patients with higher frequencies of exhausted HBV-specific CD8 T cells.ConclusionHigher levels of functionally exhausted HBV-specific CD8 T cells are associated with a lack of response that cannot be restored by blocking the PD-1:PD-L1 axis. This suggests that the clinical effectiveness of blocking the PD-1:PD-L1 axis as a monotherapy may be restricted. Combination strategies, potentially including the combination of anti-LAG-3 with other anti-iR antibodies, will likely be required to elicit a functional cure for patients with high levels of functionally exhausted HBV-specific CD8 T cells.

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PTPN11 Mutated Acute Myeloid Leukemia (AML) Features an Abundant Epichaperome Network and Is Sensitive to the Epichaperome Inhibitor PU-H71

Blood ◽

10.1182/blood-2019-128276 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 3935-3935

Author(s):

Justin D. Kaner ◽

Montreh Tavakkoli ◽

Lisa Toudic ◽

Brett M. Stevens ◽

Craig T Jordan ◽

...

Keyword(s):

Flow Cytometry ◽

T Cells ◽

Board Of Directors ◽

Peripheral Blood ◽

Research Funding ◽

Cellular Proliferation ◽

Ex Vivo ◽

Nature Medicine ◽

Advisory Board ◽

Advisory Committees

PTPN11 mutations occur in roughly 5-10% of AML patients. There is no defined impact on prognosis in the literature, although a single center analysis from our group suggested an association with poor outcomes. The pathogenesis of activating PTPN11 mutations is hypothesized to result in increased signaling through RAS resulting in uncontrolled cellular proliferation. PU-H71 is an epichaperome inhibitor that has shown pre-clinical promise in ex-vivo and PDX models of AML (Cell Reports 2015). The efficacy of the drug is dependent on the tumor possessing epichaperomes, hyperconnected networks of chaperones and co-chaperones facilitating tumor survival (Nature 2016, Nature Medicine 2018). Prior studies have found an association between epichaperome abundance and overactivity of the transcription factor MYC. Activating PTPN11 mutations have also been shown to result in MYC overactivity through increased SHP2 signaling (protein product of PTPN11 gene) and thus we hypothesized that through increased activity of MYC, activating PTPN11 mutations in AML lead to an abundant epichaperome network, and in turn render these tumors sensitive to the epichaperome inhibitor, PU-H71. 15 PTPN11 mutated AML peripheral blood and bone marrow samples from two academic medical centers (Weill-Cornell Medical Center and University of Colorado) were collected to be analyzed for epichaperome abundance and sensitivity to the epichaperome inhibitor PU-H71. To detect epichaperome abundance, PTPN11 mutated AML samples were incubated with FITC-bound PU-H71 (labeled F2) as well as a chemically altered FITC- bound PU-H71 "control" (labeled F9) which does not bind the epichaperome (Bioorg Med Chem Lett 2011). The AML cell line MV411 was used as a positive control (given known epichaperome abundance and sensitivity to PU-H71) and intra-sample T-cells were used as negative controls. These samples were analyzed using multi-parameter flow cytometry including markers to distinguish blasts, progenitors and lymphocytes. For analysis of sensitivity to PU-H71, samples were incubated with two different concentrations of PU-H71, 1uM and 0.5uM, for 48 hours and subsequently evaluated for viability using multi-parameter flow cytometry (including markers to distinguish blasts, progenitors and lymphocytes). Leukemic blasts and T cells were gated based on immunophenotype, and the ratio of blast F2 to F9 was combined with the F2-F9 blast to T-cell ratio to calculate epichaperome abundance, using a cut-off value of 4.5 to determine medium to high or low levels of the epichaperome (medium to high, >/=4.5, low, <4.5). Of the 15 samples analyzed for epichaperome abundance. Seven out of 15 (46.7%) samples showed an abundant epichaperome. Nine of these samples had complete viability data, of which, five out of nine (55.5%) showed >60% cell death after 48 hour incubation with PU-H71. For these nine samples with data on sensitivity to PU-H71, the presence or absence of epichaperome abundance was predictive of ex vivo sensitivity (5 sensitive, 4 resistant). We have established PDX-mouse models to evaluate in vivo sensitivity to PU-H71, using epichaperome abundant samples. We have confirmed the epichaperome levels in AML blast cells from the peripheral blood of the animals. Currently the treatment and analysis is ongoing. These data suggest that PTPN11 mutated AML represents a subset of AML that could be particularly sensitive to epichaperome inhibition. Additionally, the findings from our studies suggest this assay has promise as a means to accurately predict sensitivity to PU-H71, a useful precision medicine tool for a difficult to treat disease. All samples are currently being analyzed for MYC transcriptional activity and other signaling pathways. Disclosures Chiosis: Samus Therapeutics: Equity Ownership, Patents & Royalties: Intellectual rights to the PU-FITC assay. Ritchie:agios: Other: Advisory board; Pfizer: Other: Advisory board, travel support; Celgene: Other: Advisory board; Celgene, Novartis: Other: travel support; Celgene, Incyte, Novartis, Pfizer: Consultancy; Ariad, Celgene, Incyte, Novartis: Speakers Bureau; AStella, Bristol-Myers Squibb, Novartis, NS Pharma, Pfizer: Research Funding; Tolero: Other: Advisory board; Genentech: Other: Advisory board; Jazz Pharmaceuticals: Research Funding. Desai:Astellas: Honoraria; Celgene: Consultancy; Cellerant: Consultancy; Astex: Research Funding; Sanofi: Consultancy. Lee:Ai Therapeutics: Research Funding; Karyopharm Therapeutics: Consultancy; AstraZeneca Pharmaceuticals: Consultancy; Roche Molecular Systems: Consultancy; Helsinn: Consultancy; Jazz Pharmaceuticals, Inc: Consultancy. Roboz:Astex: Consultancy, Membership on an entity's Board of Directors or advisory committees; Argenx: Consultancy, Membership on an entity's Board of Directors or advisory committees; Amphivena: Consultancy, Membership on an entity's Board of Directors or advisory committees; Agios: Consultancy, Membership on an entity's Board of Directors or advisory committees; Actinium: Consultancy, Membership on an entity's Board of Directors or advisory committees; AbbVie: Consultancy, Membership on an entity's Board of Directors or advisory committees; Trovagene: Consultancy, Membership on an entity's Board of Directors or advisory committees; Takeda: Consultancy, Membership on an entity's Board of Directors or advisory committees; Sandoz: Consultancy, Membership on an entity's Board of Directors or advisory committees; Roche/Genentech: Consultancy, Membership on an entity's Board of Directors or advisory committees; Pfizer: Consultancy, Membership on an entity's Board of Directors or advisory committees; Otsuka: Consultancy, Membership on an entity's Board of Directors or advisory committees; Orsenix: Consultancy, Membership on an entity's Board of Directors or advisory committees; MEI Pharma: Consultancy, Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees; Jazz: Consultancy, Membership on an entity's Board of Directors or advisory committees; Janssen: Consultancy, Membership on an entity's Board of Directors or advisory committees; Eisai: Consultancy, Membership on an entity's Board of Directors or advisory committees; Daiichi Sankyo: Consultancy, Membership on an entity's Board of Directors or advisory committees; Celltrion: Consultancy, Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees; Bayer: Consultancy, Membership on an entity's Board of Directors or advisory committees; Astellas: Consultancy, Membership on an entity's Board of Directors or advisory committees. Guzman:Cellectis: Research Funding; SeqRx: Consultancy; Samus Therapeutics: Patents & Royalties: intellectual rights to the PU-FITC assay.

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588 Targeting GITR enhances human tumour-infiltrating T cell functionality in mismatch repair proficient primary colorectal carcinoma and liver metastases

Journal for ImmunoTherapy of Cancer ◽

10.1136/jitc-2020-sitc2020.0588 ◽

2020 ◽

Vol 8 (Suppl 3) ◽

pp. A623-A623

Author(s):

Yannick Rakké ◽

Lucia Campos Carrascosa ◽

Adriaan van Beek ◽

Valeska de Ruiter ◽

Michael Doukas ◽

...

Keyword(s):

Flow Cytometry ◽

T Cells ◽

T Cell ◽

Colorectal Carcinoma ◽

Liver Metastases ◽

Mismatch Repair ◽

Immune Checkpoint ◽

Ex Vivo ◽

Human Tumour ◽

Functional Assays

BackgroundImmune checkpoint blockade (ICB; e.g. anti-PD-1/-CTLA-4) has been proven to be clinically effective in mismatch repair deficient (dMMR) colorectal carcinoma (CRC). Yet, the majority of patients carry mismatch repair proficient (pMMR) CRC, especially those with liver metastasis, and do not respond to ICB. Here, we studied the effect of immune checkpoint stimulation via GITR targeting on human tumour-infiltrating lymphocyte (TIL) functionality in pMMR primary CRC and liver metastases (CRLM).MethodsHuman TIL were isolated from freshly resected pMMR tumours of patients with primary CRC (stage 1–3) or liver metastases (table 1). GITR expression on TIL was determined using flow cytometry and compared to leukocytes isolated from blood (PBMC) and tumour-free surrounding tissues (tumour-free colon/liver, resp. TFC and TFL). Ex vivo functional assays were used to assess TIL expansion, activation and cytokine/cytotoxic mediator secretion upon CD3/CD28 bead activation and co-stimulation using an antibody-crosslinked recombinant trimeric GITR ligand (GITRL).ResultsGITR was overexpressed on TIL when compared to other stimulatory immune checkpoints (4-1BB, OX40). GITR expression was enhanced on CD4+ and CD8+ TIL compared to PBMC and TFC or TFL compartments in both primary CRC and CRLM. Among CD4+ TIL, GITR was increasingly expressed on CD45RA± FoxP3- helper T (Th), CD45RA- FoxP3int activated helper T (aTh), and CD45RA- FoxP3hi activated regulatory T cells (aTreg), respectively. Within CD8+ TIL, GITR expression was higher on TOX+ PD1Hi and putatively tumour-reactive CD103+ CD39+ TIL.1 Impaired effector cytokine production upon ex vivo PMA/ionomycin stimulation was observed in CD4+ and CD8+ GITR-expressing TIL, hinting to functional exhaustion of the target population. However, recombinant GITRL reinvigorated ex vivo TIL responses by significantly enhancing CD4+ and CD8+ TIL numbers and proinflammatory cytokine secretion in a dose-dependent manner (figure 1). Treg depletion did not fully abrogate the stimulatory effect of GITR ligation on CD4+ and CD8+ T cell expansion, demonstrating that the stimulatory effect was partly exerted via direct targeting GITR on effector T cells. Importantly, GITR-ligation also enhanced expansion of purified CD8+CD39+ TIL. Dual treatment with GITR ligand and nivolumab (anti-PD-1) further enhanced CD8+ TIL responses compared to GITR ligand monotherapy, whereas nivolumab alone did not show any effect.Abstract 588 Table 1Patient characteristicsPatient characteristics of patients included for FACS analysis and/or functional assays. † Pathologic staging was performed according to the AJCC 8th edition criteriaAbstract 588 Figure 1GITR ligation enhances CD4+ and CD8+ TIL expansionTIL were isolated from CRC or CRLM and cultured upon CD3/CD28 activation with or without GITRL (0.1–1.0 ug/mL) for 8 days. TIL numbers were acquired by flow cytometry and normalized to counting beads. Indicated is fold change relative to ctrl-treated TIL (n=10).ConclusionsAgonistic targeting of GITR enhances ex vivo human TIL functionality in pMMR CRC and might therefore be a promising approach for novel mono- or combinatorial immunotherapies in primary CRC and CRLM.AcknowledgementsN/ATrial RegistrationN/AEthics ApprovalThe study was approved by the medical ethics committee of the Erasmus Medical Center (MEC-2012-331).ConsentN/AReferenceDuhen T, Duhen R, Montler R, et al. Co-expression of CD39 and CD103 identifies tumor-reactive CD8 T cells in human solid tumors. Nat Commun 2018;9(1):2724. doi: 10.1038/s41467-018-05072-0.

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FOXP3 expression accurately defines the population of intratumoral regulatory T cells that selectively accumulate in metastatic melanoma lesions

Blood ◽

10.1182/blood-2008-06-163048 ◽

2008 ◽

Vol 112 (13) ◽

pp. 4953-4960 ◽

Cited By ~ 98

Author(s):

Mojgan Ahmadzadeh ◽

Aloisio Felipe-Silva ◽

Bianca Heemskerk ◽

Daniel J. Powell ◽

John R. Wunderlich ◽

...

Keyword(s):

T Cells ◽

Tumor Microenvironment ◽

Metastatic Melanoma ◽

Treg Cell ◽

Peripheral Blood ◽

Ex Vivo ◽

Production Capacity ◽

Biological Marker ◽

Foxp3 Expression ◽

Treg Cells

Abstract Regulatory T (Treg) cells are often found in human tumors; however, their functional characteristics have been difficult to evaluate due to low cell numbers and the inability to adequately distinguish between activated and Treg cell populations. Using a novel approach, we examined the intracellular cytokine production capacity of tumor-infiltrating T cells in the single-cell suspensions of enzymatically digested tumors to differentiate Treg cells from effector T cells. Similar to Treg cells in the peripheral blood of healthy individuals, tumor-infiltrating FOXP3+CD4 T cells, unlike FOXP3− T cells, were unable to produce IL-2 and IFN-γ upon ex vivo stimulation, indicating that FOXP3 expression is a valid biological marker for human Treg cells even in the tumor microenvironment. Accordingly, we enumerated FOXP3+CD4 Treg cells in intratumoral and peritumoral sections of metastatic melanoma tumors and found a significant increase in proportion of FOXP3+CD4 Treg cells in the intratumoral compared with peritumoral areas. Moreover, their frequencies were 3- to 5-fold higher in tumors than in peripheral blood from the same patients or healthy donors, respectively. These findings demonstrate that the tumor-infiltrating CD4 Treg cell population is accurately depicted by FOXP3 expression, they selectively accumulate in tumors, and their frequency in peripheral blood does not properly reflect tumor microenvironment.

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Comprehensive Evaluation of the Effects of Long-term Cryopreservation on Peripheral Blood Mononuclear Cells using Flow Cytometry

10.21203/rs.3.rs-1059931/v1 ◽

2021 ◽

Author(s):

Bo Li ◽

Chunmei Yang ◽

Gui Ja ◽

Yansheng Liu ◽

Na Wang ◽

...

Keyword(s):

Flow Cytometry ◽

T Cells ◽

Peripheral Blood Mononuclear Cells ◽

Peripheral Blood ◽

Mononuclear Cells ◽

Peripheral Blood Mononuclear ◽

Effective Utilization ◽

Blood Mononuclear Cells ◽

Important Evidence

Abstract Human peripheral blood mononuclear cells (PBMCs) originate from hematopoietic stem cells (HSCs) in the bone marrow, which mainly includes lymphocytes (T cells, B cells, and natural killer [NK] cells) and monocytes. Cryopreserved PBMCs providing biobank resources are crucial for clinical application or scientific research. Here, we used flow cytometry to explore the influence of long-term cryopreservation on the quality of PBMCs with the aim of providing important evidence for the effective utilization of biobank resources. The PBMCs were isolated from the peripheral blood, which was collected from volunteers in the hospital. After long-term cryopreservation in liquid nitrogen, we analyzed the changes in cell numbers, viability, and multiple subtypes of PBMCs and studied the apoptosis, proliferation, activation, function, and status of T cells in comparison with freshly isolated PBMCs by flow cytometry, and then further tracked the effects of long-term cryopreservation on the same sample. Although the different cell types in the PBMCs dynamically changed compared with those in the freshly isolated samples, PBMC recovery and viability remained stable after long-term cryopreservation, and the number of most innate immune cells (e.g., monocytes and B cells) was significantly reduced compared to that of the freshly isolated PBMCs or long-term cryopreserved PBMCs; more importantly, the proportion of T cell subtypes, apoptosis, proliferation, and functional T cells, except for Tregs, were not affected by long-term cryopreservation. However, the proportions of activated T, naïve T, central memory T, effector T, and effector memory T cells dynamically changed after long-term cryopreservation. This article provides important evidence for the effective utilization of biobank resources. Long-term cryopreserved PBMCs can be partly used as biological resources for clinical research or basic studies, but the effect of cryopreservation on PBMCs should be considered when selecting cell samples, especially in research relating to activating or inhibiting function.

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Cytokine production in ex-vivo stimulated fresh and cryopreserved T-cells

Acta Marisiensis - Seria Medica ◽

10.2478/amma-2021-0012 ◽

2021 ◽

Vol 67 (2) ◽

pp. 95-101

Author(s):

Monica Vuță ◽

Ionela-Maria Cotoi ◽

Ion Bogdan Mănescu ◽

Doina Ramona Manu ◽

Minodora Dobreanu

Keyword(s):

T Cells ◽

Peripheral Blood ◽

Cd4 T Cells ◽

Mononuclear Cells ◽

Cytokine Production ◽

Ex Vivo ◽

Gradient Centrifugation ◽

Intracellular Cytokine Staining ◽

Interleukin 2 ◽

Middle Aged

Abstract Objective: In vitro cytokine production by peripheral blood mononuclear cells (PBMCs) is an important and reliable measure of immunocompetence. PBMC can be stimulated directly after isolation or frozen for later use. However, cryopreservation may affect cell recovery, viability and functionality. This study aims to investigate cytokine synthesis in ex-vivo stimulated fresh and cryopreserved CD4+ and CD4- T cells. Methods: PBMCs were obtained by Ficoll gradient centrifugation from heparinized peripheral blood of 6 middle-aged clinically healthy subjects. Half of these cells (labeled “Fresh”) was further processed and the other half (labeled “Cryo”) was cryopreserved at -140°C for up to 3 months. Fresh-PBMCs were activated with Phorbol-Myristate-Acetate/Ionomycin/Monensin for 5 hours immediately after isolation while Cryo-PBMCs were identically activated after thawing and cell resting. Activated cells were fixed, permeabilized and intracellular cytokine staining was performed using Phycoerythrin (PE)-conjugated antibodies for Interleukin-2 (IL-2), Tumor Necrosis Factor-alpha (TNF-a), and Interferon-gamma (IFN-g). All samples were analyzed within 24 hours by flow cytometry. Results: Both Fresh and Cryo CD3+CD4+/CD3+CD4- sub-populations partially produced each of the three cytokines. A higher percentage of CD4+ T cells produced IL-2 and TNF-a and a greater percentage of CD4- T cells were found to produce IFN-g. A significantly higher percentage of Cryo-lymphocytes was shown to produce TNF-a in both CD3+CD4+ (31.4% vs 24.9%, p=0.031) and CD3+CD4- (22.7% vs 17.9%, p=0.031) subpopulations. No notable difference was found for IL-2 and IFN-g production between Fresh and Cryo T cells. Conclusion: Cryopreservation for up to 3 months significantly increases TNF-a production of T-cells in clinically healthy middle-aged subjects.

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Persistent High Frequencies of Varicella-Zoster Virus ORF4 Protein-Specific CD4+ T Cells after Primary Infection

Journal of Virology ◽

10.1128/jvi.00564-06 ◽

2006 ◽

Vol 80 (19) ◽

pp. 9772-9778 ◽

Cited By ~ 27

Author(s):

Louise Jones ◽

Antony P. Black ◽

Gathsaurie N. Malavige ◽

Graham S. Ogg

Keyword(s):

T Cells ◽

Varicella Zoster Virus ◽

Peripheral Blood ◽

Primary Infection ◽

Ex Vivo ◽

High Frequencies ◽

Varicella Zoster ◽

Early Protein ◽

Orf4 Protein ◽

Ifn Γ

ABSTRACT Open reading frame 4 (ORF4) of varicella-zoster virus (VZV) encodes an immediate-early protein that is believed to be important for viral infectivity and establishing latency. Evidence suggests that VZV-specific T cells are crucial in the control of viral replication, but there are no data addressing the existence of potential ORF4 protein-specific CD4+ T cells. We tested the hypothesis that VZV ORF4 protein-specific CD4+ T cells could be identified and characterized within the peripheral blood of healthy immune donors following primary infection. Gamma interferon (IFN-γ) immunosorbent assays were used to screen peripheral blood mononuclear cells obtained from healthy seropositive donors for responses to overlapping ORF4 peptides, viral lysate, and live vaccine. High frequencies of ORF4 protein-specific T cells were detected ex vivo in individuals up to 52 years after primary infection. Several immunogenic regions of the ORF4 protein were identified, including a commonly recognized epitope which was restricted through HLA-DRB1*07. Total ORF4 protein-specific responses comprised 19.7% and 20.7% of the total lysate and vaccine responses, respectively, and were dominated by CD4+ T cells. Indeed, CD4+ T cells were found to dominate the overall virus-specific IFN-γ cellular immune response both ex vivo and after expansion in vitro. In summary, we have identified an ORF4 protein as a novel target antigen for persistent VZV-specific CD4+ T cells, with implications for disease pathogenesis and future vaccine development.

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Expression of CD10 by Human T Cells That Undergo Apoptosis Both In Vitro and In Vivo

Blood ◽

10.1182/blood.v94.9.3067 ◽

1999 ◽

Vol 94 (9) ◽

pp. 3067-3076 ◽

Cited By ~ 48

Author(s):

Giovanna Cutrona ◽

Nicolò Leanza ◽

Massimo Ulivi ◽

Giovanni Melioli ◽

Vito L. Burgio ◽

...

Keyword(s):

T Cells ◽

Peripheral Blood ◽

Ex Vivo ◽

Lymphoid Cells ◽

Normal Peripheral Blood ◽

Apoptotic Process ◽

Cd10 Expression ◽

T Cell Lines

Abstract This study shows that human postthymic T cells express CD10 when undergoing apoptosis, irrespective of the signal responsible for initiating the apoptotic process. Cells from continuous T-cell lines did not normally express CD10, but became CD10+ when induced into apoptosis by human immunodeficiency virus (HIV) infection and exposure to CD95 monoclonal antibody, etoposide, or staurosporin. Inhibitors of caspases blocked apoptosis and CD10 expression. Both CD4+ and CD8+ T cells purified from normal peripheral blood expressed CD10 on apoptotic induction. CD10 was newly synthesized by the apoptosing cells because its expression was inhibited by exposure to cycloheximide and CD10 mRNA became detectable by reverse transcription-polymerase chain reaction in T cells cultured under conditions favoring apoptosis. To show CD10 on T cells apoptosing in vivo, lymph node and peripheral blood T cells from HIV+ subjects were used. These suspensions were composed of a substantial, although variable, proportion of apoptosing T cells that consistently expressed CD10. In contrast, CD10+ as well as spontaneously apoptosing T cells were virtually absent in peripheral blood from normal individuals. Collectively, these observations indicate that CD10 may represent a reliable marker for identifying and isolating apoptosing T cells in vitro and ex vivo and possibly suggest novel functions for surface CD10 in the apoptotic process of lymphoid cells.

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Intracellular Cytokine Detection by Flow Cytometry in Surface Marker‐Defined Human Peripheral Blood Mononuclear T Cells

Current Protocols in Toxicology ◽

10.1002/cptx.26 ◽

2017 ◽

Vol 73 (1) ◽

Cited By ~ 3

Author(s):

Fredine T. Lauer ◽

Jesse L. Denson ◽

Ellen Beswick ◽

Scott W. Burchiel

Keyword(s):

Flow Cytometry ◽

T Cells ◽

Peripheral Blood ◽

Human Peripheral Blood ◽

Surface Marker ◽

Intracellular Cytokine ◽

Peripheral Blood Mononuclear ◽

Cytokine Detection

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3236 Identification of exhaustive markers in cytotoxic T-cells to guide immune modulation in hepatocellular carcinoma ex vivo

Journal of Clinical and Translational Science ◽

10.1017/cts.2019.33 ◽

2019 ◽

Vol 3 (s1) ◽

pp. 13-13

Author(s):

Lauren Norell Krumeich ◽

Tatiana Akimova ◽

Jason Stadanlick ◽

Abhishek Rao ◽

Neil Sullivan ◽

...

Keyword(s):

Flow Cytometry ◽

T Cells ◽

Cd8 T Cells ◽

Tumor Response ◽

Checkpoint Inhibitors ◽

Ex Vivo ◽

Receptor Expression ◽

Inhibitory Receptors ◽

Surface Receptors ◽

Study Population

OBJECTIVES/SPECIFIC AIMS: Objective: apply checkpoint inhibitors that are specific to the exhaustive markers expressed on tumor CD8+ T-cells ex vivo in order to improve cytokine release and cytotoxic function in comparison to two control groups: (1.) T-cells that receive no antibodies; (2.) T-cells that receive standard inhibition with PD-1 and CTLA-4 antibodies only. Long-term objective: provide personalized medicine in the treatment of HCC by using checkpoint inhibitors that are specific to the receptors expressed by an individual tumor. METHODS/STUDY POPULATION: The study population includes patients undergoing liver transplantation or surgical resection for HCC. Two grams of tumor, two grams of healthy liver tissue at least one centimeter from the tumor margin, and 50 milliliters of blood will be obtained. Solid tissue will be mechanically and enzymatically disrupted and CD8+ T-cells will be isolated from all sites. Using flow cytometry, the expression of surface receptors PD-1, CTLA-4, LAG-3, TIM-3, BTLA, CD244, and CD160 will be categorized in each tissue to identify which receptors are upregulated in the tumor microenvironment. Up to three antibodies specific to the upregulated receptor(s) on the tumor T-cells will be applied per specimen. The experimental arm will receive these antibodies and co-stimulation with CD3/CD28 and will be compared to two controls. One control will receive only CD3/CD28, and the other will receive CD3/CD28 in addition to the standard combination of PD-1 and CTLA-4 inhibitors. From each condition, flow cytometry will be used to assess the mean production of interleukin-2, tumor necrosis factor-α, interferon-γ, granzyme B, and perforin expression as an assessment of T-cell function. RESULTS/ANTICIPATED RESULTS: Preliminary data from the peripheral blood of healthy controls confirms that the developed flow cytometry panels effectively identify the surface receptors and cytokine production of CD8+ T-cells. Two patients have successfully been enrolled in this study. It is predicted that T-cells extracted from the tumor will express more inhibitory receptors than normal liver or peripheral blood and will have increased function after they are targeted with checkpoint inhibitors that are specific to the inhibitory surface receptors they express. DISCUSSION/SIGNIFICANCE OF IMPACT: HCC is the second leading cause of cancer-related death worldwide and therapeutic options are limited for patients who are not surgical candidates. T-cells are a critical component of the anti-tumor response to HCC. However, T-cells can develop an exhausted phenotype characterized by up-regulated inhibitory receptors (PD-1, CTLA-4, LAG-3, TIM-3, CD-244, CD-160, BTLA) and decreased function, allowing for immune escape. Clinical trials using combined checkpoint inhibition with PD-L1 and CTLA-4 antibodies have been considered a breakthrough for patients with advanced HCC, as up to 25% show an objective tumor response. The explanation for the varied susceptibility to checkpoint inhibition remains unknown and is hypothesized to be secondary to inconsistencies in the expression of surface inhibitory receptors. Although inhibitory receptor expression has been shown to be upregulated under conditions of hepatitis and/or HCC, there has been no single study to effectively investigate the expression of all known inhibitors in order to better explore the interplay between them. It will be of great academic interest and clinical purpose to evaluate individual receptor expression and engage the correlating antibodies given the possibility of synergism between receptors and the need for a more profound anti-tumor T-cell response in HCC.

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