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.

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.

scholarly journals Lactobacillus plantarum surface-displayed ASFV (p54) with porcine IL-21 generally stimulates protective immune responses in mice

AMB Express ◽  
2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Xiao-Lei Chen ◽  
Jun-Hong Wang ◽  
Wei Zhao ◽  
Chun-Wei Shi ◽  
Kai-Dian Yang ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
T Cells ◽  
Immune Responses ◽  
Fusion Protein ◽  
Lactobacillus Plantarum ◽  
Cd8 T Cells ◽  
Classical Swine Fever ◽  
Oral Vaccines ◽  
Ifn Γ ◽  
Mtt Assays

AbstractAfrican classical swine fever virus (ASFV) has spread seriously around the world and has dealt with a heavy blow to the pig breeding industry due to the lack of vaccines. In this study, we produced recombinant Lactobacillus plantarum (L. plantarum) expressing an ASFV p54 and porcine IL-21 (pIL-21) fusion protein and evaluated the immune effect of NC8-pSIP409-pgsA'-p54-pIL-21 in a mouse model. First, we verified that the ASFV p54 protein and p54-pIL-21 fusion protein were anchored on the surface of L. plantarum NC8 by flow cytometry, immunofluorescence and Western blotting. Then, the results were verified by flow cytometry, ELISA and MTT assays. Mouse-specific humoral immunity and mucosal and T cell-mediated immune responses were induced by recombinant L. plantarum. The results of feeding mice recombinant L. plantarum showed that the levels of serum IgG and mucosal secreted IgA (SIgA), the number of CD4 and CD8 T cells, and the expression of IFN-γ in CD4 and CD8 T cells increased significantly, and lymphocyte proliferation occurred under stimulation with the ASFV p54 protein. Our data lay a foundation for the development of oral vaccines against ASFV in the future.

scholarly journals 4-1BB co-stimulation further enhances anti-PD-1-mediated reinvigoration of exhausted CD39+ CD8 T cells from primary and metastatic sites of epithelial ovarian cancers

2020 ◽  
Vol 8 (2) ◽  
pp. e001650 ◽  
Author(s):  
Galam Leem ◽  
Junsik Park ◽  
Minwoo Jeon ◽  
Eui-Soon Kim ◽  
Sang Wun Kim ◽  
...  
Keyword(s):  
Ovarian Cancer ◽  
T Cells ◽  
T Cell ◽  
T Cell Activation ◽  
Cd8 T Cells ◽  
Peripheral Blood ◽  
Functional Restoration ◽  
Ovarian Cancers ◽  
Metastatic Sites ◽  
Activation Status

BackgroundResponses to immunotherapy vary between different cancer types and sites. Here, we aimed to investigate features of exhaustion and activation in tumor-infiltrating CD8 T cells at both the primary and metastatic sites in epithelial ovarian cancer.MethodsTumor tissues and peripheral blood were obtained from 65 patients with ovarian cancer. From these samples, we isolated tumor-infiltrating lymphocytes (TILs) and peripheral blood mononuclear cells. These cells were used for immunophenotype using multicolor flow cytometry, gene expression profile using RNA sequencing and ex vivo functional restoration assays.ResultsWe found that CD39+ CD8 TILs were enriched with tumor-specific CD8 TILs, and that the activation status of these cells was determined by the differential programmed cell death protein 1 (PD-1) expression level. CD39+ CD8 TILs with high PD-1 expression (PD-1high) exhibited features of highly tumor-reactive and terminally exhausted phenotypes. Notably, PD-1high CD39+ CD8 TILs showed similar characteristics in terms of T-cell exhaustion and activation between the primary and metastatic sites. Among co-stimulatory receptors, 4-1BB was exclusively overexpressed in CD39+ CD8 TILs, especially on PD-1high cells, and 4-1BB-expressing cells displayed immunophenotypes indicating higher degrees of T-cell activation and proliferation, and less exhaustion, compared with cells not expressing 4-1BB. Importantly, 4-1BB agonistic antibodies further enhanced the anti-PD-1-mediated reinvigoration of exhausted CD8 TILs from both primary and metastatic sites.ConclusionSeverely exhausted PD-1high CD39+ CD8 TILs displayed a distinctly heterogeneous exhaustion and activation status determined by differential 4-1BB expression levels, providing rationale and evidence for immunotherapies targeting co-stimulatory receptor 4-1BB in ovarian cancers.

scholarly journals CD137 Agonist Induces Gastric Cancer Cell Apoptosis by Enhancing The Functions of CD8+ T Cells via NF-κB Signaling

2020 ◽  
Author(s):  
Ben-Shun Hu ◽  
Tian Tang ◽  
Tie-Long Wu ◽  
Ying-Yue Sheng ◽  
Yu-Zheng Xue
Keyword(s):  
Gastric Cancer ◽  
Flow Cytometry ◽  
T Cells ◽  
Real Time ◽  
Peripheral Blood ◽  
Cell Apoptosis ◽  
Nuclear Translocation ◽  
Granzyme B ◽  
Ifn Γ

Abstract Background: CD137 is identified as a target for tumor immunotherapy. However, the role of CD137 in gastric cancer (GC), especially in inducing GC cell apoptosis has not been studied yet. Methods: Foxp3+ and CD8+ T cells in GCs were investigated by immunohistochemistry (IHC). CD137 expression in GCs was detected by flow cytometry, IHC and immunofluorescence (IF). Peripheral blood mononuclear cells (PBMCs) and CD8+ T cells isolated from peripheral blood were stimulated with a CD137 agonist in vitro. CD8+ T cells proliferation and p65 expression were explored by flow cytometry. p65 nuclear translocation was analyzed by IF. IL-10, TGF-β, IFN-γ, Perforin and Granzyme B were detected by real-time quantitative PCR (real-time PCR). PBMCs and primary GC cells were cocultured and stimulated with the CD137 agonist in vitro. Apoptosis of the primary GC cells was detected by flow cytometry. Results: Our data demonstrated that GC tumors show characteristics of an immunosuppressive microenvironment. CD137 was predominantly expressed in CD8+ T cells in GCs and had a positive correlation with tumor cell differentiation. CD137 agonist promoted CD8+ T cells proliferation and increased the secretion of IFN-γ, Perforin and Granzyme B, which induced primary GC cell apoptosis. Mechanistically, this study found that CD137 agonist could induce NF-κB nuclear translocation in CD8+ T cells. Conclusion: Our results demonstrate that CD137 agonist can induce primary GC cell apoptosis by enhancing CD8+ T cells via activating NF-κB signaling.

LncRNA-ENST00000421645 promotes T cells to secrete IFN-γ by sponging PCM-1 in neurosyphilis

Epigenomics ◽  
2021 ◽  
Author(s):  
Wen-Na Liu ◽  
Kai-Xuan Wu ◽  
Xiao-Tong Wang ◽  
Li-Rong Lin ◽  
Man-Li Tong ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
T Cells ◽  
Peripheral Blood ◽  
Long Noncoding Rna ◽  
Noncoding Rna ◽  
Chip Assay ◽  
Rna Immunoprecipitation ◽  
Ifn Γ ◽  
Significant Expression

Aim: Neurosyphilis patients exhibited significant expression of long noncoding RNA (lncRNA) in peripheral blood T lymphocytes. In this study, we further clarified the role of lncRNA- ENST00000421645 in the pathogenic mechanism of neurosyphilis. Methods: lncRNA- ENST00000421645 was transfected into Jurkat-E6-1 cells, namely lentivirus (Lv)-1645 cells. RNA pull-down assay, flow cytometry, RT-qPCR, ELISA (Neobioscience Technology Co Ltd, Shenzhen, China) and RNA immunoprecipitation chip assay were used to analyze the function of lncRNA- ENST00000421645. Results: The expression of IFN-γ in Lv-1645 cells was significantly increased compared to that in Jurkat-E6-1 cells stimulated by phorbol-12-myristate-13-acetate (PMA). Then, it was suggested that lncRNA- ENST00000421645 interacts with PCM1 protein. Silencing PCM1 significantly increased the level of IFN-γ in Lv-1645 cells stimulated by PMA. Conclusion: This study revealed that lncRNA- ENST00000421645 mediates the production of IFN-γ by sponging PCM1 protein after PMA stimulation.

PBMC-06- Intracellular Cytokine Staining (ICS) of IFN-gamma, IL-4 and IL-17 on Human Peripheral Blood Mononuclear Cells (PBMC) v1

2021 ◽  
Author(s):  
Alessia Furgiuele ◽  
Emanuela Rasini ◽  
Maria Giulia Albizzati ◽  
Alessandra Luini ◽  
Marco Ferrari ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
T Cells ◽  
Peripheral Blood Mononuclear Cells ◽  
Peripheral Blood ◽  
Mononuclear Cells ◽  
Brefeldin A ◽  
Intracellular Cytokine ◽  
Peripheral Blood Mononuclear ◽  
Blood Mononuclear Cells ◽  
Ifn Γ

This present protocol is developed to analyze the frequency of IFN-γ-, IL-4- and IL-17-producing CD4+T cells, identified from ex vivo human peripheral blood mononuclear cells (PBMC). The frequencies of cytokine producing cells derived from activation of PBMC was induced trough the stimulus phorbol 12-myristate 13-acetate (PMA) and ionomycin. According onpreviously published protocols concentrations of stimulating substances were in the range from 10, to 50 ng/ml for PMA and 1 µg/ml for ionomycin (Gupta and Maecker, 2015; Foster et al., 2007; Freer and Rindi, 2013; https://www.bdbiosciences.com/content/bdb/paths/generate-tds-document.in.560751.pdf). The PMA concentrations of 10, 20 and 50 ng/ml were tested and finally the PMA concentration of 10 ng/ml was chosen since it was sufficient to obtain a frequency of cytokines comparable to that obtained with higher stimulus concentrations. PMA/ionomycin and brefeldin A are incubate together for a time of 5 h (Gupta and Maecker, 2015, Foster et al., 2007, Freer and Rindi, 2013, https://www.bdbiosciences.com/content/bdb/paths/generate-tds-document.in.560751.pdf). The protein secretion inhibitor brefeldin A, was used at the concentration of 10 µg/ml (Gupta and Maecker, 2015; Foster et al., 2007; Freer and Rindi, 2013). Cell concentrations may vary in a range from 2.5 x106 to 10 x106 cells/ml (Maecker, 2004; Freer and Rindi, 2013a; https://www.bdbiosciences.com/content/bdb/paths/generate-tds-document.in.560751.pdf). Concentration of 1x106 cells/ml, 4x106 cells/ml and 8x106cells/ml were tested. Cell tritation have shown a higher functional response proportional to the cell concentration when exposed to a fixed concentration of stimulants. Cell concentration of 8 milions/ml was selected in order to obtain the higher percentage of IFN-γ-, IL-4- and IL-17-producing CD4+T cells. In conclusion the present protocol provides that, for a optimal optimal percentage of IFN-γ-, IL-4- and IL-17-producing CD4+T cells as assessed by flow cytometry (Table 1), PBMC in a concentration 8 milions/ml were stimulated with PMA 10 ng/ml and ionomycin 1 µg/ml, and cultured for 5 h in presence of brefeldin A 10 µg/ml according to the procedure described in detail below. References Baran, J., Kowalczyk, D., Ozog, M., Zembala, M., 2001. Three-color flow cytometry detection of intracellular cytokines in peripheral blood mononuclear cells: Comparative analysis of phorbol myristate acetate-ionomycin and phytohemagglutinin stimulation. Clin. Diagn. Lab. Immunol. 8, 303–313. https://doi.org/10.1128/CDLI.8.2.303-313.2001 Foster, B., Prussin, C., Liu, F., Whitmire, J.K., Whitton, J.L., 2007. Detection of intracellular cytokines by flow cytometry. Curr. Protoc. Immunol. Chapter 6. https://doi.org/10.1002/0471142735.im0624s78 Freer, G., Rindi, L., 2013. Intracellular cytokine detection by fluorescence-activated flow cytometry: Basic principles and recent advances. Methods 61, 30–38. https://doi.org/10.1016/j.ymeth.2013.03.035 Gupta, S., Maecker, H., 2015. Intracellular Cytokine Staining (ICS) on Human Lymphocytes or Peripheral Blood Mononuclear Cells (PBMCs). BIO-PROTOCOL 5. https://doi.org/10.21769/bioprotoc.1442 Maecker, H.T., 2004. Cytokine flow cytometry. Methods Mol. Biol. 263, 95–108. https://doi.org/10.1385/1-59259-773-4:095 https://www.bdbiosciences.com/content/bdb/paths/generate-tds-document.us.560751.pdf BEFORE STARTING with this procedure Moreover, work under laminar flow hood when you are processing samples from the beginning to the end of the culture. Make sure you are using, sterile culture medium and sterile plastic disposable as well.

Efficacy of Immunomodulatory Therapy with All-Trans Retinoid Acid for Adults with Chronic Immune Thrombocytopenia

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 3335-3335
Author(s):  
Lan Dai ◽  
Zhaoyue Wang ◽  
Ri Zhang ◽  
Xia Bai ◽  
Mingqing Zhu ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
T Cells ◽  
Peripheral Blood ◽  
Immune Thrombocytopenia ◽  
Pcr Analysis ◽  
Therapeutic Effects ◽  
Rt Pcr ◽  
Platelet Destruction ◽  
Prednisone Treatment ◽  
Ifn Γ

Abstract Abstract 3335 Objective: Immune thrombocytopenia (ITP) is an immune-mediated disorder characterized by antibody- and cell-mediated platelet destruction and impaired platelet production. In addition of antibody-mediated platelet clearance, T-cell is also involved in platelet destruction or marrow suppression. During immune responses CD4+ T cells can differentiate into a range of cell types, including T helper type 1 (Th1), Th2, Th17 and regulatory T cells (Treg). The goal of the present study is to investigate the therapeutic effects of all-trans-retinoic acid (ATRA) plus prednisone treatment on adult refractory idiopathic thrombocytopenic purpura (RITP) and to further explore the underlying mechanisms. Methods: All 35 patients were treated with ATRA at fixed dose of 10 mg/tid plus prednisone and were started after discontinuation of other previous ITP treatment. Peripheral blood samples were collected from 35 RITP patients and 20 healthy subjects. The concentrations of the peripheral blood CD 4+ T cells (Th1, Th2, TH17, TREG) were analyzed by flow cytometry, the levels of cytokines were confirmed by enzyme-linked immunoassay (ELISA), and the expression of T-bet, GATA-3, RORγt, FOXP3 were detected by semiquantitative reverse-transcription polymerase chain reaction (RT-PCR) in 35 RITP patients before and after treatments, with 20 normal individuals as respective controls. Results: The overall response rate of ATRA plus prednisone treatment in RITP patients was 54.3%, 28.6%(n=10) of patients with a complete response and 25.7%(n=9) of patients with a partial response. Among the 19 responders, the mean platelet count was 34+13×106/ml before ATRA therapy and 106+29×106/ml after treatment. No severe adverse effects happened. The levels of regulory T cells were significantly increased in patients after ATRA plus prednisone treatment (P < 0.05); the levels of Th1, Th2, Th17 were not significantly improved (P >0.05) in effective patients after treatments in flow cytometry analysis. The concentrations of IL-10, TGF-β was increased (P<0.05), whereas IFN-γ, IL-4, IL-17 factor showed no obvious change in the effective groups after treatment (P > 0.05). The expression levels of Foxp3 enhanced dramatically after treatment in real-time RT-PCR analysis (P < 0.05). However, the concentrations of T-bet, GATA-3 and ROR-γ showed no obvious change after the therapy in real-time RT-PCR analysis(P > 0.05). Conclusions: 54.3% of RITP patients recovered after ATRA plus prednisone treatments. The therapeutic effects of ATRA plus prednisone may be involved in the increased levels of regulory T cells in peripheral blood through increased expression of TGF-β, IL-10, and Foxp3. In comparison, the therapeutic effects may not be attributed to the expression of TH1, TH2, TH17, IFN-γ, IL-4, IL-17, T-bet, GATA-3, RORγt. Disclosures: No relevant conflicts of interest to declare.

Determination of CD4+ and CD8+ T cells in the peripheral blood of dogs with demodicosis

Parasitology ◽  
2010 ◽  
Vol 137 (13) ◽  
pp. 1921-1924 ◽  
Author(s):  
S. K. SINGH ◽  
U. DIMRI ◽  
M. C. SHARMA ◽  
B. SHARMA ◽  
M. SAXENA
Keyword(s):  
Flow Cytometry ◽  
T Cells ◽  
Cd8 T Cells ◽  
Peripheral Blood ◽  
Cd4 T Cells ◽  
Significant Alteration ◽  
Healthy Controls ◽  
Healthy Dogs

SUMMARYThe aim of this study was to evaluate the CD4+/CD8+ ratio in peripheral blood of dogs with localized and generalized demodicosis. Sixteen dogs were examined, 8 with localized and 8 with generalized demodicosis, while 8 healthy dogs were used as controls. Peripheral blood was obtained and CD4+ and CD8+ T cells were determined by flow cytometry. Significantly higher numbers of CD8+ T cells and lower numbers of CD4+ T cells were found in dogs with generalized demodicosis compared to dogs with localized demodicosis and healthy controls. Significantly higher numbers of CD8+ T cells and lower numbers of CD4+ T cells were also found in dogs with localized demodicosis compared to healthy controls. The CD4+/CD8+ ratio was also found to be significantly lower in dogs with generalized demodicosis in comparison with dogs with localized demodicosis and healthy controls. It is concluded that significant alteration in the CD4+/CD8+ ratio may be implicated in the pathogenesis of generalized canine demodicosis.

scholarly journals The profile of leucocytes, CD3+, CD4+, and CD8+ T cells, and cytokine concentrations in peripheral blood of children with acute asthma exacerbation

2017 ◽  
Vol 45 (6) ◽  
pp. 1658-1669 ◽  
Author(s):  
Thuy Nguyen-Thi-Dieu ◽  
Huong Le-Thi-Thu ◽  
Sy Duong-Quy
Keyword(s):  
Flow Cytometry ◽  
T Cells ◽  
Cd8 T Cells ◽  
Peripheral Blood ◽  
Asthma Exacerbation ◽  
Acute Asthma ◽  
Gm Csf ◽  
Paediatric Patients ◽  
Tnf Α ◽  
Acute Asthma Exacerbation

Objective To determine the leucocyte profile and cytokine concentrations in the peripheral blood of children with an acute asthma exacerbation (AAE). Methods This descriptive, cross-sectional study enrolled paediatric patients admitted to hospital for AAE. The severity of AAE was assessed using the paediatric asthma score (PAS). Peripheral blood samples were collected for automatic quantification of white blood cell counts, CD3+, CD4+, and CD8+ T cells populations by flow cytometry and cytokine concentrations by flow cytometry-assisted immunoassay. Results A total of 127 children with AAE and 30 healthy control subjects were included in the study. The proportion of paediatric patients with decreased CD3+, CD4+ and CD8+ T cells was significantly higher in those with severe AAE compared with those with mild-to-moderate AAE. The concentrations of interleukin (IL)-2, IL-8, IL-12, and IL-4 in paediatric patients with rhinovirus infection were significantly higher than in those without rhinovirus infection. IL-2, IL-4, IL-6, TNF-α and GM-CSF concentrations during AAE were significantly lower than control. IL-5 and IL-13 concentrations during AAE were significantly higher than control. Conclusions The decrease of CD3+, CD4+, CD8+ T cells and IL-2, IL-4, IL-6, TNF-α, and GM-CSF combined with the increase of IL-5 and IL-13, were associated with AAE in children with asthma.

scholarly journals 234 Distinct efficacy and immunological responses to αPD-1, αPD-L1 and αPD-L2 immunotherapy in aged versus young hosts

2021 ◽  
Vol 9 (Suppl 3) ◽  
pp. A249-A250
Author(s):  
Yilun Deng ◽  
Harshita Gupta ◽  
Myrna Garcia ◽  
Aravind Kancharla ◽  
Ryan Reyes ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cancer Immunotherapy ◽  
Cd8 T Cells ◽  
Immune Checkpoint ◽  
Checkpoint Blockade ◽  
List Type ◽  
Aged Mice ◽  
Melanoma Model ◽  
Ifn Γ

BackgroundAging is the biggest risk factor for cancer, yet there are limited pre-clinical/clinical data regarding aging effects on immune checkpoint (IC) inhibition (ICI) outcomes. αPD-1 can potentially block PD-L1 and PD-L2 while αPD-L1 can block PD-1 and CD80. Melanoma response to αPD-1/αPD-L1 correlates with CD8+TCF-1+ T cell stem cell (TCSC) generation.1 Lack of host IL-17 can lead to increased IFN-γ production.2 3MethodsWe tested αPD-1 (200 μg/mouse), αPD-L1 (100 μg/mouse) or αPD-L2 (200 μg/mouse) in aged (18–33 months) and young (3–8 months) mice challenged orthotopically with B16 (WT or PD-L1ko) or TPN61R melanoma (NRAS mutation melanoma model)4 (αPD-L2 only) (SQ). Tumors were analyzed by flow. We tested αPD-L2 (20 μg/ml) effects by co-culturing young or aged T cells ± young or aged myeloid cells.ResultsWe reported that αPD-1 treats young and aged with B16 whereas αPD-L1 treats young not aged.5 αPD-L2 treated B16 and TPN61R melanoma in aged but, remarkably, not young, the first single agent anti-cancer immunotherapy exhibiting this property (figure 1). B16 tumors from aged had differential IC content (PD-1, PD-L1, CD80, PD-L2) versus tumors from young (e.g., more PD-L2+ tumor and stroma cells in aged mice; figure 2). Efficacy in young (αPD-1, αPD-L1) and aged (αPD-L2) correlated with increased tumor TCSC content (figure 3). αPD-L2 efficacy against B16 in aged mice required host IFN-γ and IL-17 (figure 4). αPD-1 efficacy against B16 in aged appeared to be host and tumor PD-L1 independent (figure 5). PD-L1KO B16 response to αPD-1 in aged also correlated with increased tumor TCSC content. Myeloid cell PD-L2 signaling inhibited aged but not young CD8+ T cell IL-2 production in vitro (figure 6).Abstract 234 Figure 1Abstract 234 Figure 2Abstract 234 Figure 3Abstract 234 Figure 4Abstract 234 Figure 5Abstract 234 Figure 6ConclusionsTreatment differences in aged versus young could depend on IC, TCSC and/or host cytokine differences (IL-17/IFN-γ). αPD-1 efficacy in aged PD-L1KO mice challenged with PD-L1KO B16 suggests that PD-L2 block is sufficient for αPD-1 efficacy in aged. PD-L2 expression differences in the tumor microenvironment could also contribute to treatment efficacy differences. PD-L2 inhibitory signaling on aged but not young CD8+ T cells is a likely mechanism for αPD-L2 efficacy in aged but not young. We are now testing the role of IL-17 in αPD-L2 efficacy as it could be upstream of IFN-γ effects, and TCSC effects in aged versus young. Our work can improve cancer immunotherapy in aged hosts and provides insights into treatment failure, including in young hosts.AcknowledgementsSouth Texas MSTP training grant (NIH T32GM113896), TL1TR002647, NIH T32AI138944, R01 CA231325, Waxman Grant, UL1 TR001120ReferencesMiller BC, Sen DR, Al Abosy R, Bi K, Virkud YV, LaFleur MW, Yates KB, Lako A, Felt K, Naik GS, et al. Subsets of exhausted CD8(+) T cells differentially mediate tumor control and respond to checkpoint blockade. Nat Immunol 2019;20(3):326–336.Moroda M, Takamoto M, Iwakura Y, Nakayama J, Aosai F. Interleukin-17A-deficient mice are highly susceptible to toxoplasma gondii infection due to excessively induced T. gondii HSP70 and interferon gamma production. Infection and immunity 2017;85(12):e00399–00317.Yi T, Zhao D, Lin C-L, Zhang C, Chen Y, Todorov I, LeBon T, Kandeel F, Forman S, Zeng D. Absence of donor Th17 leads to augmented Th1 differentiation and exacerbated acute graft-versus-host disease. Blood, The Journal of the American Society of Hematology 2008;112(5):2101–2110.Burd CE, Liu W, Huynh MV, Waqas MA, Gillahan JE, Clark KS, Fu K, Martin BL, Jeck WR, Souroullas GP. Mutation-specific RAS oncogenicity explains NRAS codon 61 selection in melanoma. Cancer discovery 2014;4(12):1418–1429.Padron A, Hurez V, Gupta HB, Clark CA, Pandeswara SL, Yuan B, Svatek RS, Turk MJ, Drerup JM, Li R, et al. Age effects of distinct immune checkpoint blockade treatments in a mouse melanoma model. Exp Gerontol 2018;105:146–154.Ethics ApprovalAll animal studies are approved by UTHSA IACUC.

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