High-Dimensional Flow Cytometry Analysis of Regulatory Receptors on Human T Cells, NK Cells, and NKT Cells

Helper T cells are known to mediate hepatic ischemia/reperfusion (I/R) injury. However, the precise mechanisms and subsets of CD4+ T cells that contribute to this injury are still controversial. Therefore, we sought to determine the contributions of different CD4+ T cell subsets during hepatic I/R injury. Wild-type, OT-II, or T cell receptor (TCR)-δ-deficient mice were subjected to 90 min of partial hepatic ischemia followed by 8 h of reperfusion. Additionally, wild-type mice were pretreated with anti-CD1d, -NK1.1, or -IL-2R-α antibodies before I/R injury. OT-II mice had diminished liver injury compared with wild-type mice, implicating that antigen-dependent activation of CD4+ T cells through TCRs is involved in hepatic I/R injury. TCR-δ knockout mice had decreased hepatic neutrophil accumulation, suggesting that γδ T cells regulate neutrophil recruitment. We found that natural killer T (NKT) cells, but not NK cells, contribute to hepatic I/R injury via CD1d-dependent activation of their TCRs, as depletion of NKT cells by anti-CD1d antibody or depletion of both NKT cells and NK cells by anti-NK1.1 attenuated liver injury. Although regulatory T cells (Treg) are known to suppress T cell-dependent inflammation, depletion of Treg cells had little effect on hepatic I/R injury. The data suggest that antigen-dependent activation of CD4+ T cells contributes to hepatic I/R injury. Among the subsets of CD4+ T cells, it appears that γδ T cells contribute to neutrophil recruitment and that NKT cells directly injure the liver. In contrast, NK cells and Treg have little effects on hepatic I/R injury.

Download Full-text

PD-1 expression on NK cells can be related to cytokine stimulation and tissue residency

10.1101/2021.03.29.437486 ◽

2021 ◽

Author(s):

Arnika K Wagner ◽

Nadir Kadri ◽

Chris Tibbitt ◽

Koen van de Ven ◽

Sunitha Bagawath-Singh ◽

...

Keyword(s):

Flow Cytometry ◽

T Cells ◽

Nk Cells ◽

Mhc Class I ◽

Sequencing Analysis ◽

Single Cell Rna Sequencing ◽

Cytokine Stimulation ◽

Deficient Mice

ABSTRACTAlthough PD-1 was shown to be a hallmark of T cells exhaustion, controversial studies have been reported on the role of PD-1 on NK cells. Here, we found by flow cytometry and single cell RNA sequencing analysis that PD-1 can be expressed on MHC class I-deficient tumor-infiltrating NK cells in vivo. We also demonstrate distinct alterations in the phenotype of PD-1-deficient NK cells which in part could be attributed to a decrease in tumor-infiltrating NK cells in PD-1-deficient mice. NK cells from PD-1-deficient mice exhibited a more mature phenotype which might reduce their capacity to migrate and kill in vivo. Finally, our results demonstrate that PD-L1 molecules in membranes of PD-1-deficient NK cells migrate faster than in NK cells from wildtype mice, suggesting that PD-1 and PD-L1 form cis interactions with each other on NK cells.

Download Full-text

Cisplatin Increased the Expression of PD-1 and PD-L1 by YAP1 in Hepatocellular Carcinoma

10.21203/rs.3.rs-763998/v1 ◽

2021 ◽

Author(s):

Liyuan Hao ◽

Yinglin Guo ◽

Qing Peng ◽

Zhiqin Zhang ◽

Shenghao Li ◽

...

Keyword(s):

Hepatocellular Carcinoma ◽

Flow Cytometry ◽

T Cells ◽

Cancer Cells ◽

Flow Cytometry Analysis ◽

Chemotherapy Drugs ◽

The World ◽

Hcc Cells

Abstract Hepatocellular carcinoma (HCC) was one of the most malignant cancers in the world. Cisplatin (DDP) was one of the main chemotherapy drugs for HCC, but the mechanism of DDP treatment for HCC remains unclear. In this presentation, we found that DDP inhibited the growth of HCC cells and promoted the expression of PD-1 and its ligand PD-L1 in cancer cells. Meanwhile, flow cytometry analysis revealed that DDP enhanced PD-1-CD8+ T cells expression and decreased PD-1+CD8+ T cells expression. ELISA analysis suggested that DDP decreased TGF-β expression in vivo. Therefore, the study indicated that DDP enhanced PD-1 and PD-L1 expression and inhibited the growth of HCC.

Download Full-text

High dimensional flow cytometry for comprehensive leukocyte immunophenotyping (CLIP) in translational research

Journal of Immunological Methods ◽

10.1016/j.jim.2010.06.010 ◽

2011 ◽

Vol 363 (2) ◽

pp. 245-261 ◽

Cited By ~ 26

Author(s):

Angélique Biancotto ◽

John C. Fuchs ◽

Ann Williams ◽

Pradeep K. Dagur ◽

J. Philip McCoy

Keyword(s):

Flow Cytometry ◽

Translational Research ◽

High Dimensional ◽

Dimensional Flow

Download Full-text

Identification of Small-Molecule Inducers of FOXP3 in Human T Cells Using High-Throughput Flow Cytometry

Single Cell Analysis - Series in BioEngineering ◽

10.1007/978-981-10-4499-1_11 ◽

2017 ◽

pp. 243-252 ◽

Cited By ~ 1

Author(s):

Rob Jepras ◽

Poonam Shah ◽

Metul Patel ◽

Steve Ludbrook ◽

Gregory Wands ◽

...

Keyword(s):

Flow Cytometry ◽

T Cells ◽

Small Molecule ◽

High Throughput ◽

Human T Cells

Download Full-text

Sinonasal T-Cell Expression of Cytotoxic Mediators Granzyme B and Perforin is Reduced in Patients with Chronic Rhinosinusitis

American Journal of Rhinology and Allergy ◽

10.2500/ajra.2017.31.4474 ◽

2017 ◽

Vol 31 (6) ◽

pp. 352-356 ◽

Cited By ~ 1

Author(s):

Sarah E. Smith ◽

Rodney J. Schlosser ◽

James R. Yawn ◽

Jose L. Mattos ◽

Zachary M. Soler ◽

...

Keyword(s):

Flow Cytometry ◽

T Cells ◽

T Cell ◽

Nk Cells ◽

Chronic Rhinosinusitis ◽

Nasal Polyp ◽

Granzyme B ◽

Cytotoxic Cells ◽

Sinonasal Mucosa ◽

Perforin Expression

Background CD8+ T cells and natural killer (NK) cells are cytotoxic cells that use granzyme B (GrB) and perforin. Defective cytotoxic function is known to play a role in dysregulated immune response as seen in chronic sinusitis, also referred to as chronic rhinosinusitis (CRS). However, to our knowledge, in the United States, neither GrB or perforin expression has been reported in patients with CRS. Objective The aim of this study was to investigate sinonasal cytotoxic cells, their mediators, and cell-specific distribution of these mediators in patients with CRS with nasal polyp (CRSwNP) and in patients with CRS without nasal polyp (CRSsNP). Methods Blood and sinus tissue samples were taken from patients with CRSsNP (n = 8) and CRSwNP (n = 8) at the time of surgery. Control subjects (n = 8) underwent surgery for cerebrospinal fluid leak repair or to remove non-hormone-secreting pituitary tumors. The cells were analyzed via flow cytometry by using CD8 expression to identify cytotoxic T cells and CD56 expression to identify NK cells. Intracellular GrB and perforin expression were analyzed with flow cytometry. Results We observed no significant differences in plasma or peripheral blood immune cell numbers or specific levels of GrB or perforin among the groups. In the sinonasal mucosa of the patients with CRSsNP and the patients with CRSwNP, there was a significant decrease in GrB and perforin levels (p <0.05) despite similar or increased numbers of cytotoxic cells when compared with the controls. The overall decrease in GrB and perforin in the sinonasal mucosa of the patients with CRSsNP and the patients with CRSwNP was due to decreased T cell production. There was no difference in total NK cell count or expression of perforin or GrB among all the groups. Conclusion Total levels of sinonasal GrB and perforin were decreased in the sinonasal mucosa of both the patients with CRSwNP and the patients with CRSsNP compared with the controls, whereas sinonasal CD8+ T cells, (but not NK cells,), intracellular stores of GrB and perforin were reduced in the patients with CRSwNP compared with the controls.

Download Full-text

Flow Cytometry and FTIR Spectroscopy for Detection of Early Apoptosis in Human T Cells

Proceedings ◽

10.3390/proceedings2251558 ◽

2018 ◽

Vol 2 (25) ◽

pp. 1558 ◽

Cited By ~ 1

Author(s):

Günnur Güler ◽

Ayten Nalbant

Keyword(s):

Flow Cytometry ◽

T Cells ◽

Cell Death ◽

Ftir Spectroscopy ◽

Apoptotic Cell ◽

Annexin V ◽

Apoptotic Cell Death ◽

Biochemical Changes ◽

Human T Cells ◽

Early Apoptosis

Apoptosis, a programmed cell death, has a vital role in various cellular processes. Apoptotic cells exhibit morphological and biochemical changes, detected by a variety of assays (caspases, mitochondrial dyes, DNA laddering). Flow cytometry is a powerful tool for detection of apoptotic cell death and allows information about the cell size and molecules associated with cell-bound antibodies. Recently, Fourier transform infrared (FTIR) spectroscopy as rapid and low-cost tool has been extensively used for cellular studies, providing information on cellular structures. The aim of this study was to detect early apoptosis and obtain further insights into the capability of FTIR spectroscopy, comparing the results with flow cytometry. In this study, apoptotic cell death was induced in human Jurkat T cells with Camptothecin (CPT), a DNA topoisomerase I inhibitor. Cells were cultured with 4µM CPT in RPMI (with 5% FCS) for 24 h. Immunoflourescence labeling for multicolor flow cytometry was accomplished with Annexin V concomitantly with 7-AAD. The same cells were also analyzed with ATR-FTIR spectroscopy. Flow cytometry data represents that the cells are Annexin V positive but 7AAD negative. This indicates that cells are in the early apoptotic stage, only externalization of phosphatidylserine exists on the plasma membrane. FTIR data reveals that membrane phospholipids and proteins undergo changes; fatty acid acyl chains are disordered and increased in mobility after treatment, which result from the early apoptosis process after CPT-treatment, confirmed by the flow cytometry. A combined study of flow cytometry and FTIR spectroscopy for analysis of apoptosis in human T cells exhibited compatible and complementary results. Existence of biophysical and biochemical changes in T cells after treatment were also demonstrated.

Download Full-text

B7-H4 Inhibits the Development of Primary Sjögren’s Syndrome by Regulating Treg Differentiation in NOD/Ltj Mice

Journal of Immunology Research ◽

10.1155/2020/4896727 ◽

2020 ◽

Vol 2020 ◽

pp. 1-13

Author(s):

Xu Zheng ◽

Qikai Wang ◽

Xiang Yuan ◽

Yingbo Zhou ◽

Hui Chu ◽

...

Keyword(s):

Flow Cytometry ◽

T Cells ◽

Salivary Gland ◽

T Cell ◽

Salivary Glands ◽

Inflammatory Cytokines ◽

Mrna Levels ◽

Lymphocyte Infiltration ◽

Flow Cytometry Analysis ◽

Treg Differentiation

Background. This study is aimed at exploring the role of B7-H4 in the pathogenesis of primary Sjögren’s syndrome (pSS) in NOD/Ltj mouse. Methods. B7-H4 expression in salivary glands was examined by IHC, and lymphocyte infiltration was showed by H&E. Next, anti-B7-H4 mAb or immunoglobulin isotype was injected into NOD/Ltj mice. Cytokine levels were measured by quantitative RT-PCR, and immunoglobulins were measured by ELISA. T cell subsets were analyzed by flow cytometry. Last, we treated NOD/Ltj mice with B7-H4Ig and control Ig. CD4+Foxp3+ T cells were assessed by immunohistochemistry. Two-tailed Student’s t-tests were used to detect the statistical difference in various measures between the two groups. Results. B7-H4 expression was remarkably reduced in salivary glands of NOD/Ltj mice at 15 weeks compared with the NOD/Ltj mice at 8 weeks. Anti-B7-H4 mAb treatment increased lymphocyte infiltration in salivary glands. Inflammatory cytokines including IL-12, IL-18, IL-1α, TNF-α, IFN-α, and BAFF were upregulated markedly in anti-B7-H4 mAb-treated mice compared to IgG isotype-treated mice. Flow cytometry analysis showed that anti-B7-H4 mAb-treated mice had lower levels of CD4+Foxp3+/CD4+ T cells in spleen. Moreover, Foxp3 mRNA levels of salivary glands were diminished in anti-B7-H4 mAb-treated mice. Flow cytometry analysis showed that anti-B7-H4 mAb inhibited CD4+Foxp3+/CD4+ T cell production, while B7-H4Ig would promote naïve CD4+ T into Treg differentiation. Administration with B7-H4Ig displayed significantly decreased lymphocyte infiltration in salivary glands and low levels of total IgM and IgG in serum. Analysis of inflammatory cytokines in salivary glands after B7-H4Ig treatment revealed that the mRNA levels of IL-12, IL-6, IL-18, IL-1α, TNF-α, and IFN-α were significantly downregulated in B7-H4Ig-treated mice compared to control Ig treatment. B7-H4Ig-treated mice had significantly higher levels of CD4+Foxp3+/CD4+ T cells in spleen. IHC in salivary gland revealed that CD4+Foxp3+ T cells of B7-H4Ig treatment mouse were more than control Ig treatment. Conclusions. Our findings implicate that B7-H4 has a protective role for salivary gland epithelial cells (SGECs) and therapeutic potential in the treatment of pSS.

Download Full-text