OMIP-020: Phenotypic characterization of human γδ T-cells by multicolor flow cytometry

2014 ◽  
Vol 85 (6) ◽  
pp. 522-524 ◽  
Author(s):  
Kilian Wistuba-Hamprecht ◽  
Graham Pawelec ◽  
Evelyna Derhovanessian
Keyword(s):  
Flow Cytometry ◽  
T Cells ◽  
Γδ T Cells ◽  
Phenotypic Characterization ◽  
Multicolor Flow Cytometry

838 Phenotypic and functional signatures of peripheral and tumor-resident γδ T cells are informative for outcome of checkpoint blockade in melanoma

2020 ◽  
Vol 8 (Suppl 3) ◽  
pp. A890-A890
Author(s):  
Graham Pawelec ◽  
Kilian Wistuba-Hamprecht ◽  
Kilian Wistuba-Hamprecht
Keyword(s):  
Flow Cytometry ◽  
T Cells ◽  
Replicative Senescence ◽  
Γδ T Cells ◽  
Cytokine Expression ◽  
Checkpoint Blockade ◽  
Phenotypic Characterization ◽  
List Type ◽  
Gammadelta T Cells

BackgroundImmune checkpoint blockade (ICB) set a milestone in cancer immunotherapy, but still only a fraction of patients responds. Thus, there is an urgent need for biomarkers predicting outcome, and also for understanding the responsible mechanisms. γδ T cells constitute a numerically minor subset of 1-10% of the peripheral T cell compartment in healthy people and have a major role in defense against multiple microbial and non-microbial challenges. Unlike the majority of T cells, γδ T cells bind their ligands in an MHC-independent manner. We previously studied γδ T cells, that also express checkpoint molecules, in patients in the pre-checkpoint blockade era and thereafter, and identified correlations between subset frequencies of these unconventional T cells and patients‘ overall survival (OS). Here, we present a detailed phenotyping and functional investigation of tumor-resident as well as peripheral γδ T cells.MethodsPhenotyping was performed in stage IV melanoma patients before and under PD-1+/-CTLA-4 blockade using as basis our published OMIP-20 protocol.1 Cytokine expression patterns and proliferative capacities were determined as described according to our established protocols.2 Primary flow cytometry data analysis was performed using FlowJo (BD) and correlations with clinical meta data were determined using Prism (GraphPad) and SPSS (IBM).ResultsWe found previously that low frequencies of peripheral Vδ1 γδ T cells were associated with prolonged OS. Here, we investigated functional aspects and abundance of γδ T cells within the tumor as well as in the blood. The peripheral Vδ1 but not the Vδ2 differentiation signature revealed significantly lower proportions of naive and effector cells as well as an accumulation of late differentiated cells in patients with high Vδ1 frequencies. The cytokine expression pattern (IFNγ, TNF and IL-17) and the degranulation marker CD107a were different in patients with high versus low peripheral Vδ1 frequencies. The proliferative capabilities of Vδ1 cells in melanoma were limited in comparison to healthy subjects. Both Vδ1 and Vδ2 cells were found in tumor tissues, and these analyses are ongoing, including analyses of replicative senescence through CD57 expression.ConclusionsOur data provide novel insights into the role of γδ T cells in cancer rejection. The previously found negative correlation of Vδ1 T cells with OS is likely due to an accumulation of mal-functioning, probably exhausted Vδ1 T cells in patients with poor outcome of ICB. Thus, we suggest that Vδ1 T cells are promising candidates for future exploitation in novel ICB-approaches.Ethics ApprovalThis study was approved by K. Wistuba-Hamprecht´s Ethics Committee (approval nos. 490/2014BO1 and 792/2016BO2).ReferencesWistuba-Hamprecht K, Pawelec G, Derhovanessian E. OMIP-020: Phenotypic characterization of human gammadelta T-cells by multicolor flow cytometry. Cytometry A 2014;85:522–524. doi:10.1002/cyto.a.22470Beucke N, et al. Pitfalls in the characterization of circulating and tissue-resident human gammadelta T cells. J Leukoc Biol 2020. doi:10.1002/JLB.5MA1219-296R

Phenotypic characterization of γδ T cells mobilized in response to acute psychological stress

2010 ◽  
Vol 24 (4) ◽  
pp. 608-614 ◽  
Author(s):  
Leila H. Anane ◽  
Kate M. Edwards ◽  
Victoria E. Burns ◽  
Jet J.C.S. Veldhuijzen van Zanten ◽  
Mark T. Drayson ◽  
...  
Keyword(s):  
T Cells ◽  
Psychological Stress ◽  
Γδ T Cells ◽  
Phenotypic Characterization ◽  
Acute Psychological Stress

Identification and phenotypic characterization of γδ T cells in rat lymph

2012 ◽  
Vol 93 (1) ◽  
pp. 168-171 ◽  
Author(s):  
Sudhanshu Shekhar ◽  
Simon Milling ◽  
Chris Jenkins ◽  
Gordon MacPherson
Keyword(s):  
T Cells ◽  
Γδ T Cells ◽  
Phenotypic Characterization

scholarly journals Characterization of γδ T-cells via flow cytometry

AGE ◽  
2015 ◽  
Vol 37 (6) ◽  
Author(s):  
Kilian Wistuba-Hamprecht ◽  
Graham Pawelec
Keyword(s):  
Flow Cytometry ◽  
T Cells ◽  
Γδ T Cells

Development and characterization of a HCMV multiantigen therapeutic vaccine for GBM using the UNITE platform.

2021 ◽  
Vol 39 (15_suppl) ◽  
pp. e14565-e14565
Author(s):  
Amit Adhikari ◽  
Juliete Macauley ◽  
Yoshimi Johnson ◽  
Mike Connolly ◽  
Tim Coleman ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
T Cells ◽  
T Cell ◽  
Tumor Microenvironment ◽  
Mouse Model ◽  
T Cell Activation ◽  
Cell Activation ◽  
Cd8 T Cell ◽  
T Cell Response

e14565 Background: Glioblastoma (GBM) is an aggressive form of brain cancer with a median survival of 15 months which has remained unchanged despite technological advances in the standard of care. GBM cells specifically express human cytomegalovirus (HCMV) proteins providing a unique opportunity for targeted therapy. Methods: We utilized our UNITE (UNiversal Intracellular Targeted Expression) platform to develop a multi-antigen DNA vaccine (ITI-1001) that codes for the HCMV proteins- pp65, gB and IE-1. The UNITE platform involves lysosomal targeting technology, fusing lysosome-associated protein 1 (LAMP1) with target antigens resulting in increased antigen presentation by MHC-I and II. ELISpot, flow cytometry and ELISA techniques were used to evaluate the vaccine immunogenicity and a syngeneic, orthotopic GBM mouse model that expresses HCMV proteins was used for efficacy studies. The tumor microenvironment studies were done using flow cytometry and MSD assay. Results: ITI-1001 vaccination showed a robust antigen-specific CD4 and CD8 T cell response in addition to a strong humoral response. Using GBM mouse model, therapeutic treatment of ITI-1001 vaccine resulted in ̃56% survival with subsequent long-term immunity. Investigating the tumor microenvironment showed significant CD4 T cell infiltration as well as enhanced Th1 and CD8 T cell activation. Regulatory T cells were also upregulated upon ITI-1001 vaccination and would be an attractive target to further improve this therapy. In addition, tumor burden negatively correlated with number of activated CD4 T cells (CD4 IFNγ+) reiterating the importance of CD4 activation in ITI-1001 efficacy and potentially identifying treatment responders and non-responders. Further characterization of these two groups showed high infiltration of CD3+, CD4+ and CD8+ T cells in responders compared with non- responders along with higher CD8 T cell activation. Conclusions: Thus, we show that vaccination with HCMV antigens using the ITI-1001-UNITE platform generates strong cellular and humoral immune responses, triggering significant anti-tumor activity that leads to enhanced survival in mice with GBM.

scholarly journals Pitfalls in the characterization of circulating and tissue‐resident human γδ T cells

2020 ◽  
Vol 107 (6) ◽  
pp. 1097-1105 ◽  
Author(s):  
Nicola Beucke ◽  
Daniela Wesch ◽  
Hans‐Heinrich Oberg ◽  
Christian Peters ◽  
Jonas Bochem ◽  
...  
Keyword(s):  
T Cells ◽  
Γδ T Cells

scholarly journals Phenotypic characterization of human cytolytic T lymphocytes in mixed lymphocyte culture.

1981 ◽  
Vol 153 (1) ◽  
pp. 213-218 ◽  
Author(s):  
A Moretta ◽  
M C Mingari ◽  
B F Haynes ◽  
R P Sekaly ◽  
L Moretta ◽  
...  
Keyword(s):  
T Cells ◽  
T Lymphocytes ◽  
Cell Surface ◽  
Mixed Lymphocyte Reaction ◽  
Mixed Lymphocyte Culture ◽  
Lymphocyte Culture ◽  
Phenotypic Characterization ◽  
Cytolytic T Lymphocytes ◽  
Activated T Cells

Mixed lymphocyte reaction (MLR)-activated T cells were analyzed according to the expression of various cell surface markers by the specific cytotoxic T lymphocytes (CTL) generated in the MLR. CTL were found exclusively in a population of MLR-activated T cells that lacked detectable Fc gamma R but that expressed a surface antigen recognized by the 4F2 monoclonal antibody. In contrast, CTL were found in both the Ia-positive and Ia-negative cells after MLR activation. Thus, the specific CTL generated in the allogeneic MLR can be identified and isolated by virtue of the expression of a particular cell surface marker.

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