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

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.

Effect of neoadjuvant chemotherapy on immune checkpoint expression in breast cancer.

2017 ◽  
Vol 35 (15_suppl) ◽  
pp. e12126-e12126
Author(s):  
Andrew Stiff ◽  
Christopher McQuinn ◽  
Robert Wesolowski ◽  
Maryam B. Lustberg ◽  
Raquel E. Reinbolt ◽  
...  
Keyword(s):  
Breast Cancer ◽  
T Cells ◽  
Cd8 T Cells ◽  
Cd4 T Cells ◽  
Triple Negative ◽  
Interaction Analysis ◽  
Checkpoint Inhibitors ◽  
Prospective Trial ◽  
Receptor Expression ◽  
Starting Point

e12126 Background: Even with neo-adjuvant chemotherapy (NAC) many breast cancer (BC) patients (pts) relapse, especially triple negative pts. The incorporation of checkpoint inhibitors into NAC for BC is being tested in clinical trials. How NAC affects checkpoint receptor expression is not known. Such information could aid in the rational selection of checkpoints to target during NAC. We sought to characterize changes in the frequency of circulating CD4 and CD8 T cells expressing PD1, CTLA4, LAG3, TIM3, and OX40 over the course of NAC. Methods: In this prospective trial, expression of PD-1, CTLA-4, Lag3, Tim3 and Ox40 on circulating CD4 and CD8 T cells were measured by FACS analysis in pts with operable breast cancer (BC) prior and at the end of NAC. The primary objective was to explore the association between NAC and expression levels of the immune checkpoints. Results: 1, 20 and 3 pts had clinical stage I, II, IIIA, respectively. Median age was 48. 11, 6 and 7 pts were triple negative (TN), HER2+ and hormone receptor (HR)+, respectively. Complete pathologic response rate was 45.8%. Globally CD4 T cells expressing CTLA4, Lag3, Ox40 and PD1 decreased following NAC (all p < 0.01). Conversely, CD8 T cells expressing CTLA4, Lag3 and Ox40 significantly increased (all p < 0.01). More CD8 T cells from HER2+ pts expressed Lag3 prior to therapy compared to HR+ pts (p < 0.05) with a similar trend compared to TN pts. Prior to therapy more CD8 T cells from HER2+ and TN pts expressed Tim3 compared to HR+ pts (p < 0.05 for each). Post therapy more CD4 T cells from HER2+ pts expressed PD1 compared to HR+ and TN pts (p = 0.027 and 0.018 respectively). Clinical response did not predict change in checkpoint expression. An interaction analysis revealed that HER2+ disease predicted a drop in CTLA4 CD4 T cells and a drop in Lag3 CD4 and CD8 T cells over NAC (p < 0.05). Conclusions: This analysis identified changes in checkpoint receptor expression by CD4 and CD8 T cells in BC pts after NAC. Differences in checkpoint receptor expression were found between BC subgroups. This data provides a starting point for understanding checkpoint receptor expression changes with NAC, and could help guide the selection and timing of incorporating checkpoint inhibitors in BC.

859 Inhibition of the kinase activity of hematopoietic progenitor kinase 1 enhances anti-PD-1-induced reinvigoration of human tumor-infiltrating CD8+ T cells

2020 ◽  
Vol 8 (Suppl 3) ◽  
pp. A912-A912
Author(s):  
Yongjoon Lee ◽  
Seung Hyuck Jeon ◽  
A Yeong Park ◽  
Suyeon Jo ◽  
Jinhwa Lee ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cd8 T Cells ◽  
Immune Checkpoint ◽  
Kinase Activity ◽  
Checkpoint Inhibitors ◽  
Ex Vivo ◽  
Human Tumor ◽  
Hematopoietic Progenitor ◽  
Effector Functions

BackgroundImmune checkpoint inhibitors (ICIs) including anti-CTLA-4, anti-PD-1, and anti-PD-L1 have been clinically used for the treatment of various types of cancer. However, ICIs have a limited efficacy, and it is required to develop a strategy to enhance the efficacy of ICIs. Hematopoietic progenitor kinase 1 (HPK1) was recently known to inhibit T cell receptor (TCR) signaling by targeting SLP76 thus suppress T-cell effector functions.MethodsIn the present study, we examined the expression of HPK-1 and SLP76 in tumor-infiltrating lymphocytes (TILs) obtained from renal cell carcinoma tissues, in relation with the expression of PD-1 and other immune checkpoint receptors by performing flow cytometry analysis. In addition, we examined if inhibition of the kinase activity of HPK1 by CMPD0914, that is a potent, selective and orally available HPK1 inhibitor, enhanced effector functions of tumor-infiltrating CD8+ T cells in the presence of anti-PD-1 blocking antibodies.ResultsFirst, we found that HPK1 and SLP76 are expressed in both CD8+ and CD4+ T cells including Foxp3+ regulatory T cells irrespective of PD-1 expression. Intriguingly, the expression levels of HPK1 and SLP76 were significantly higher in the PD-1bright population compared to the PD-1- or PD-1dim populations. Further characterization revealed that HPK1 and SLP76 were highly expressed in CD8+ T-cell populations expressing TOX, a transcription regulator of T-cell exhaustion, or TCF-1, a transcription factor related to progenitor-like exhausted T cells. In ex vivo functional assays, anti-PD-1 treatment increased the production of IFN-γ and TNF, and the expression of a proliferation marker, Ki-67 among tumor-infiltrating CD8+ T cells. Interestingly, the effects of anti-PD-1 treatment were further enhanced by the combination treatment with CMPD0914.ConclusionsIn summary, we demonstrated that HPK1 and SLP76 are expressed by human tumor-infiltrating T cells, particularly PD-1brightCD8+ T cells, and that anti-PD-1-induced T-cell reinvigoration is significantly enhanced by an HPK1 inhibitor, CMPD0914, rationalizing the combination of anti-PD1/PD-L1 and HPK1 inhibitors for the treatment of cancer.

A phase 1/2 study of a novel IL-2 cytokine, NKTR-214, and nivolumab in patients with select locally advanced or metastatic solid tumors.

2017 ◽  
Vol 35 (15_suppl) ◽  
pp. e14040-e14040 ◽  
Author(s):  
Adi Diab ◽  
Nizar M. Tannir ◽  
Chantale Bernatchez ◽  
Cara L. Haymaker ◽  
Salah E Bentebibel ◽  
...  
Keyword(s):  
T Cells ◽  
Tumor Microenvironment ◽  
Cd8 T Cells ◽  
Tumor Response ◽  
Immune Cell ◽  
Checkpoint Inhibitors ◽  
Phase 1 ◽  
Locally Advanced ◽  
Clinical Activity ◽  
Biomarker Data

e14040 Background: NKTR-214 is a CD-122-biased agonist that targets the IL-2 pathway and is designed to provide sustained signaling through the heterodimeric IL-2 receptor pathway (IL-2Rβɣ) to preferentially activate and expand NK and effector CD8+ T cells over T regulatory cells within the tumor microenvironment. In a phase 1 monotherapy trial, pts treated with NKTR-214 demonstrated a substantial increase in CD8+ T and NK cells within the tumor microenvironment even when pretreated with multiple prior immunotherapeutic agents (abstract submitted). Based on this biomarker data and a favorable safety profile, a trial combining NKTR-214 and nivolumab was initiated. Methods: This is an on-going phase 1/2 study of NKTR-214 plus nivolumab in Pts with either melanoma (Mel), NSCLC, renal, bladder, or TNBC. Pts who are immunotherapy naïve or checkpoint therapy relapse/refractory are being studied separately. NKTR-214 and nivolumab are administered IV on a q2w or q3w schedule. Cohort 1 received NKTR-214 0.006 mg/kg q3w with nivolumab 240 mg q2w. Blood and tumor tissue were collected to measure immune activation using flow cytometry, immunohistochemistry, T cell clonality and gene expression analyses. Results: As of February 7, 2017, 5 Pts have been treated with the combination and all Pts were naïve to checkpoint inhibitors. There have been no dose limiting toxicities, no drug-related or immune related grade 3-5 adverse events (TRAEs) and no Pts have discontinued treatment. The most common TRAEs were pruritis and rash. Radiographic scans were available for 2 Pts. On treatment, Pt 1 with Mel had a mixed radiographic response at 1st scan, a ~40% decrease in LDH and a robust tumor immune cell infiltrate at week 3 the majority being newly proliferating CD8+ T cells expressing PD-1. Pt 2 with Mel had an unconfirmed complete response per RECIST 1.1 after 6 weeks of treatment; follow up tumor response data will be presented. Conclusions: Preliminary data demonstrate that NKTR-214 and nivolumab combination therapy is well tolerated with early evidence of clinical activity. Updated safety, pharmacokinetics, tumor response and biomarker data will be presented. Clinical trial information: NCT02983045.

scholarly journals CD4 and CD8 co-receptors modulate functional avidity of CD1b-restricted T cells

2020 ◽  
Author(s):  
Charlotte A. James ◽  
Yuexin Xu ◽  
Melissa S. Aguilar ◽  
Lichen Jing ◽  
Erik D. Layton ◽  
...  
Keyword(s):  
T Cells ◽  
Mhc Class Ii ◽  
Cd8 T Cells ◽  
Ex Vivo ◽  
Antigen Recognition ◽  
Receptor Expression ◽  
Jurkat Cells ◽  
Sample Collection ◽  
Lipid Antigens ◽  
Lipid Antigen

ABSTRACTCD4 and CD8 co-receptors define distinct lineages of T cells restricted by major histocompatibility complex (MHC) Class II and I molecules, respectively. Co-receptors interact with the T cell receptor (TCR) at the surface of MHC-restricted T cells to facilitate antigen recognition, thymic selection, and functional differentiation. T cells also recognize lipid antigens presented by CD1 molecules, but the role that CD4 and CD8 play in lipid antigen recognition is unknown. We studied the effect of CD4 and CD8 on the avidity, activation, and function of T cells specific for two CD1b-presented mycobacterial lipid antigens, glucose monomycolate (GMM) and diacylated sulfoglycolipids (SGL). In a human cohort study using SGL-loaded CD1b tetramers, we discovered a hierarchy among SGL-specific T cells in which T cells expressing the CD4 or CD8 co-receptor stain with a higher tetramer mean fluorescence intensity (MFI) than CD4-CD8- T cells. To determine the role of the TCR co-receptor in lipid antigen recognition, we exogenously expressed GMM and SGL-specific TCRs in Jurkat or polyclonal T cells and quantified tetramer staining and activation thresholds. Transduced CD4+ primary T cells bound the lipid-loaded CD1b tetramer with a higher MFI than CD8+ primary T cells, and transduced CD8+ Jurkat cells bound the SGL-CD1b tetramer with higher MFI than CD4-CD8- Jurkat cells. The presence of either co-receptor also decreased the threshold for IFN-γ secretion. Further, co-receptor expression increased surface expression of CD3ε, suggesting a mechanism for increased tetramer binding and activation. Finally, we used single-cell sequencing to define the TCR repertoire and ex vivo functional profiles of SGL-specific T cells from individuals with M.tb disease. We found that CD8+ T cells specific for SGL express canonical markers associated with cytotoxic T lymphocytes, while CD4+ T cells could be classified as T regulatory or T follicular helper cells. Among SGL-specific T cells, only those expressing the CD4 co-receptor also expressed Ki67, suggesting that they were actively proliferating at the time of sample collection. Together, these data reveal that expression of CD4 and CD8 co-receptor modulates TCR avidity for lipid antigen, leading to functional diversity and differences in in vivo proliferation during M.tb disease.

Evaluating the repertoire of immune checkpoint markers expressed on peripheral and ascites CD8+ T cells in ovarian cancer.

2017 ◽  
Vol 35 (15_suppl) ◽  
pp. 5571-5571 ◽  
Author(s):  
Stephanie Gaillard ◽  
Chelsae Dumbauld ◽  
Alyssa Bilewski ◽  
Jessie A Ehrisman ◽  
Angeles Alvarez Secord ◽  
...  
Keyword(s):  
Ovarian Cancer ◽  
Flow Cytometry ◽  
T Cells ◽  
Cd8 T Cells ◽  
Immune Checkpoint ◽  
Peripheral Blood ◽  
Ascites Fluid ◽  
High Dimensional ◽  
Inhibitory Receptors ◽  
Ifn Γ

5571 Background: Understanding the immune checkpoint marker repertoire can facilitate development of therapeutic strategies to improve efficacy of immune-based therapies. We used a novel high-dimensional flow cytometry panel to determine co-expression patterns of immune checkpoint markers and effector function of CD8+ T cells from peripheral blood and ascites of patients newly diagnosed with ovarian cancer. Methods: Peripheral blood and ascites samples were collected from patients with epithelial ovarian cancer (n=8). Cells isolated from peripheral blood and ascites were used for immune profiling by multiparameter flow cytometry of 5 inhibitory receptors (PD-1, LAG-3, TIM-3, TIGIT, and BTLA) on CD8+ T cells, along with 4 functional parameters (production of each of the following: TNF-α, IFN-γ, IL-2, and upregulation of CD107a). A complementary multiplex analysis on plasma and ascites fluid was performed to quantify 14 soluble checkpoint markers. Results: The concentrations of soluble PD-1, TIM-3, LAG-3, CTLA-4, BTLA, IDO, and CD137 were increased in ascites fluid compared to plasma from patients with ovarian cancer. Ascites CD8+ T cells co-express higher levels of inhibitory receptors than peripheral CD8+ T cells. In total, CD8+ T cells in ascites retained the ability to produce effector functions at levels similar to peripheral blood. However, IFN-γ production was retained in PD-1 only expressing CD8+ T cells and decreased in CD8+ T cells co-expressing multiple receptors. Conclusions: High-dimensional flow cytometry allowed for the phenotypic and functional characterization of CD8+ T cells from ovarian cancer patients. The profile of receptor co-expression was distinct in peripheral blood compared to ascites. Collectively, our study suggests that co-expression of factors beyond PD-1 influences CD8+ T cell activity. Thus blocking PD-1 and PD-L1 alone may not be sufficient for CD8+ T cells expressing multiple inhibitory receptors.

P08.02 Harnessing T cells to target brain metastasis

2021 ◽  
Vol 23 (Supplement_2) ◽  
pp. ii26-ii26
Author(s):  
S R Gregory ◽  
C Fife ◽  
J Williams ◽  
H Carrasco Hope ◽  
T Andreou ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cd8 T Cells ◽  
Checkpoint Inhibitors ◽  
Ex Vivo ◽  
Adoptive T Cell Therapy ◽  
Activation Markers ◽  
T Cell Therapy ◽  
In Vivo Models ◽  
Human T Cells

Abstract BACKGROUND Up to 60% of melanoma patients develop brain metastases (BrM). These patients have a poor prognosis and limited treatment options. Immune checkpoint inhibitors (ICI) targeting Cytotoxic T-lymphocyte-associated protein-4 (CTLA-4) and Programmed cell death protein-1 (PD-1) have revolutionized the treatment of melanoma and their efficacy has been also demonstrated in melanoma BrM. Our group previously demonstrated that ICI (combined α-PD-1 and α-CTLA-4) enhances chemokine-dependent infiltration of cytotoxic T lymphocytes (CTLs) into melanoma BrMs in preclinical models, accompanied by upregulated expression of T cell attracting chemokines in tumours. Notably, CTLs infiltrating BrM expressed only some of the chemokine receptors (CRs) interacting with ICI-induced chemokines in BrM, providing a rationale to over-express the “missing” CRs in T cells to enhance their homing to tumours in the context of adoptive T cell therapy (ACT). MATERIALS AND METHODS OT-I cells were isolated from OT-I mice and differentiated ex vivo into effector (TEF) and memory (TCM) CD8+ T cells. Tumour infiltrating lymphocytes (TILs) from B16 tumour-bearing mice treated with ICI were isolated using magnetic beads, activated and expanded ex vivo. Expression of CRs and activation markers in ex vivo cultured T cells were quantified by qPCR and/or flow cytometry. The migration of human blood CD8+ T cells towards chemokines of interest were measured in ex vivo migration assays. RESULTS The same CRs that were missing on BrM-infiltrating CTLs in vivo models were also absent from OT-I TEF (CCR7low/CD44high/CD62Llow) and TCM (CCR7high/CD44low/CD62Lhigh) cells, as well as from TILs expanded ex vivo for use in ACT. Furthermore, we observed no increase in migration of human T cells towards chemokines interacting with the “missing” CRs in comparison to the baseline migration, suggesting that these CRs are also absent from human T cells. CONCLUSION Ex vivo expanded T cells that are used in ACT are missing several CRs that are interacting with chemokines upregulated in BrM. We hypothesise that the use of genetically engineered T cells expressing the “missing” CRs in ACT has the potential to enhance ACT efficacy in combination with ICI.

Synergy between glutamate modulation and anti–programmed cell death protein 1 immunotherapy for glioblastoma

2021 ◽  
pp. 1-10
Author(s):  
Ravi Medikonda ◽  
John Choi ◽  
Ayush Pant ◽  
Laura Saleh ◽  
Denis Routkevitch ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
T Cells ◽  
Cell Death ◽  
Programmed Cell Death ◽  
Cd8 T Cells ◽  
Immune Cell ◽  
Checkpoint Inhibitors ◽  
Treatment Strategies ◽  
Cell Death Protein

OBJECTIVE Immune checkpoint inhibitors such as anti–programmed cell death protein 1 (anti-PD-1) have shown promise for the treatment of cancers such as melanoma, but results for glioblastoma (GBM) have been disappointing thus far. It has been suggested that GBM has multiple mechanisms of immunosuppression, indicating a need for combinatorial treatment strategies. It is well understood that GBM increases glutamate in the tumor microenvironment (TME); however, the significance of this is not well understood. The authors posit that glutamate upregulation in the GBM TME is immunosuppressive. The authors utilized a novel glutamate modulator, BHV-4157, to determine synergy between glutamate modulation and the well-established anti-PD-1 immunotherapy for GBM. METHODS C57BL/6J mice were intracranially implanted with luciferase-tagged GL261 glioma cells. Mice were randomly assigned to the control, anti-PD-1, BHV-4157, or combination anti-PD-1 plus BHV-4157 treatment arms, and median overall survival was assessed. In vivo microdialysis was performed at the tumor site with administration of BHV-4157. Intratumoral immune cell populations were characterized with immunofluorescence and flow cytometry. RESULTS The BHV-4157 treatment arm demonstrated improved survival compared with the control arm (p < 0.0001). Microdialysis demonstrated that glutamate concentration in TME significantly decreased after BHV-4157 administration. Immunofluorescence and flow cytometry demonstrated increased CD4+ T cells and decreased Foxp3+ T cells in mice that received BHV-4157 treatment. No survival benefit was observed when CD4+ or CD8+ T cells were depleted in mice prior to BHV-4157 administration (p < 0.05). CONCLUSIONS In this study, the authors showed synergy between anti-PD-1 immunotherapy and glutamate modulation. The authors provide a possible mechanism for this synergistic benefit by showing that BHV-4157 relies on CD4+ and CD8+ T cells. This study sheds light on the role of excess glutamate in GBM and provides a basis for further exploring combinatorial approaches for the treatment of this disease.

scholarly journals Functional Exhaustion of HBV-Specific CD8 T Cells Impedes PD-L1 Blockade Efficacy in Chronic HBV Infection

2021 ◽  
Vol 12 ◽  
Author(s):  
Sara Ferrando-Martinez ◽  
Angie Snell Bennett ◽  
Elisabete Lino ◽  
Adam J. Gehring ◽  
Jordan Feld ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
T Cells ◽  
Cd8 T Cells ◽  
Ex Vivo ◽  
Peptide Pool ◽  
Clinical Effectiveness ◽  
T Cell Response ◽  
Functional Cure ◽  
Chronic Hbv

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.

scholarly journals 640 Stem-like CD4 T cells in cancer

2021 ◽  
Vol 9 (Suppl 3) ◽  
pp. A669-A669
Author(s):  
Maria Cardenas ◽  
Nataliya Prokhnevska ◽  
Caroline Jansen ◽  
Viraj Master ◽  
Haydn Kissick
Keyword(s):  
Flow Cytometry ◽  
T Cells ◽  
Single Cell ◽  
Cd4 T Cells ◽  
Tumor Response ◽  
Ex Vivo ◽  
University Hospital ◽  
In Vivo Studies ◽  
Endogenous Virus

BackgroundCD4 T cells can differentiate into multiple effector subsets that can mediate variable functions. In this work we aim to understand how CD4 T cells differentiate in response to tumor antigens and their respective function in the anti-tumor response.MethodsTumor tissue was collected from patients undergoing surgery at Emory University Hospital. Activated PD1+ CD45RA- tumor infiltrating CD4 T cells were sent for 10X single cell RNA-seq. Tumor samples were also processed for flow cytometry and ex vivo functional analyses. For in vivo studies, prostate cancer mouse model expressing the LCMV glycoprotein (TRAMPC1-GP) was used, as well as LCMV Armstrong infection.ResultsTo characterize the heterogeneity of CD4 T cells infiltrating kidney tumors, we performed single cell RNAseq. We found three distinct activated (PD1+ CD45RA-) CD4 T cell populations. Two effector clusters consisting of Th1-like (EOMES+) and Treg (FOXP3+) cells, and a third cluster expressing TCF1, and genes associated with stemness and survival that did not fit defined CD4 effector lineages. We further confirmed these data by flow cytometry and found the same tumor infiltrating CD4 subsets in 100 kidney cancer patients. When placed in culture under different polarization conditions, tumor TCF1+ CD4 T cells proliferated and differentiated into the Th1-like and Treg effector populations found in the tumor, in addition to other effector lineages (Th1, Tfh) given the appropriate conditions, while the Th1-like and Treg cells underwent no proliferation or phenotype changes. These data suggests that the TCF1+ CD4s act as activated unpolarized precursors to the effector subsets in the tumor. To further test this hypothesis in vivo, we adoptively transferred tumor specific (SMARTA) CD4 T cells into mice followed by TRAMPC1-GP tumor inoculation. Transferred SMARTAs activated and first acquired a TCF1+ phenotype in the TDLN prior to predominantly differentiating into Tregs in the tumor. Given their plasticity in vitro, we asked whether TCF1+ SMARTAs primed in tumors were destined to differentiate into Tregs. To test this, we transferred 4-week activated TCF1+ SMARTAs from TDLNs of TRAMPC1-GP mice into naïve mice that were immediately infected with LCMV Armstrong. We found that the transferred SMARTAs differentiated into Th1 and Tfh cells in response to the virus, similar to the endogenous virus specific CD4 T cells.ConclusionsOverall, this work shows that CD4 T cells remain in an activated phenotype in the tumor with the capacity to differentiate into non-suppressive effector lineages given the appropriate conditions that may benefit the anti-tumor response.

scholarly journals Beyond PD-1: Investigating the Therapeutic Potential of TIGIT Blockade in DLBCL

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 391-391 ◽  
Author(s):  
Nicole Sunseri ◽  
Xiufen Chen ◽  
Noemie Wald ◽  
Julie Preillon ◽  
Sonali M. Smith ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
T Cells ◽  
T Cell ◽  
B Cell ◽  
Cd8 T Cells ◽  
Research Funding ◽  
Cell Lymphoma ◽  
Ex Vivo ◽  
B Cell Lymphoma ◽  
Isotype Control

Background: The PD-1/PD-L1 axis is a dominant cancer immune escape pathway, and PD-1 blockade therapy has greatly benefited patients with select solid tumors and lymphomas. Unfortunately, anti-PD-1 monotherapy has limited efficacy against relapsed/refractory (r/r) diffuse large B cell lymphoma (DLBCL) - a disease where new therapies are needed. Because numerous inhibitory checkpoint receptors have been implicated in driving tumor-specific T cell dysfunction, we hypothesized that combinatorial checkpoint blockade therapy (CBT) would enhance the activity of PD-1-based therapy in r/r DLBCL. T cell immunoglobulin and immunoreceptor tyrosine-based inhibitory motif domain (TIGIT) is a recently identified co-inhibitory receptor expressed on dysfunctional tumor-infiltrating T cells. PD-1 and TIGIT co-blockade therapy has demonstrated impressive activity in pre-clinical solid tumor and myeloma models. However, the degree to which TIGIT is involved in mediating T cell suppression in DLBCL is not fully known. Methods: TIGIT expression on lymphoma-infiltrating T cells (LITs) from 18 fresh lymphoma samples was analyzed by flow cytometry. Multiplex IHC on tissue microarrays (TMAs) was also performed to investigate TIGIT expression in DLBCL samples. The syngeneic murine A20 B cell lymphoma model was employed to study: 1) the kinetics of TIGIT and other co-receptor expression on LITs, 2) the association of TIGIT expression with effector function among LITs, and 3) the effectiveness of anti-TIGIT mono- and combination CBT in mice with established A20 lymphomas. A20 lymphoma tumors were established in groups of Balb/c mice by subcutaneous (SQ) injections of 5 x 106 cells. Expression of TIGIT and other co-receptors in A20 LITs was examined by flow cytometry at various time points. Function of TIGIT+ LITs was assessed by examining cytokine production following ex vivo stimulation with PMA and ionomycin. To test the efficacy of TIGIT blockade, mice received intraperitoneal injections of anti-TIGIT, anti-PD-1, anti-4-1BB, or combinations of these antibodies. Treatments began once tumors reached a diameter of 10 mm and were continued every 3 days for 5 doses. Tumor growth was monitored and compared to that in A20-bearing mice treated with isotype control antibodies. In some experiments, mice that achieved complete tumor rejection following single or dual CBT were re-challenged with A20 cells to investigate immunological memory responses. Results: Across a variety of human lymphomas, flow cytometric analysis revealed that TIGIT was broadly upregulated on LITs, including regulatory T cells and conventional CD4+and CD8+ T cells (Figure A and B). TIGIT expression on LITs in DLBCL was particularly high. Nearly all TIGIT+ LITs were also PD-1+, suggesting that these receptors co-orchestrate a T cell dysfunctional state in the lymphoma environment. Multiplex immunofluorescence staining of DLBCL samples demonstrated that TIGIT was most highly expressed on CD8+ T cells and that TIGIT+ T cells tended to be localized near, and in some cases, surrounding CD20+ lymphoma cells. Consistent with observations in human lymphomas, LITs isolated from murine A20 lymphoma commonly co-expressed TIGIT and PD-1, and the degree of expression correlated directly with tumor volume. This correlation was also present for other co-receptors, including 4-1BB, TIM3, and CTLA-4. Ex vivo restimulation of A20 LITs revealed that TIGIT+ T cells produced lower levels of effector cytokines, such as TNF-α, compared with TIGIT- T cells. In mice with established A20 lymphomas, both TIGIT and PD-1 mono-blockade led to modest delays in tumor outgrowth compared with mice treated with isotype control antibodies. Strikingly, however, combined PD-1 and TIGIT blockade resulted in complete rejection of A20 lymphomas in most mice and led to significantly prolonged survival compared to mice treated with single agent CBT (Figure C). Combination TIGIT and 4-1BB CBT was also remarkably effective in driving rejection of A20 lymphomas, and led to remarkable memory responses. Conclusions: TIGIT promotes immune tolerance in the DLBCL environment. While TIGIT monotherapy has anti-lymphoma activity, combinatorial CBT incorporating anti-TIGIT antibodies drives extremely potent rejection of established lymphomas in mice. These results provide rationale for further study of TIGIT blockade as a therapeutic strategy in r/r lymphomas, including DLBCL. Disclosures Sunseri: iTeos Therapeutics: Research Funding. Wald:iTeos Therapeutics: Employment. Preillon:iTeos Therapeutics: Employment. Smith:Portola Pharmaceuticals: Research Funding. Driessens:iTeos Therapeutics: Employment. Kline:Merck: Honoraria; Merck: Research Funding.

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