Integrin Activation Enables Sensitive Detection of Functional CD4+ and CD8+ T Cells: Application to Characterize SARS-CoV-2 Immunity

Anna Schöllhorn; Juliane Schuhmacher; Luciana Besedovsky; Rolf Fendel; Anja T.R. Jensen; Stefan Stevanović; Tanja Lange; Hans Georg Rammensee; Jan Born; Cécile Gouttefangeas; Stoyan Dimitrov

doi:10.3389/fimmu.2021.626308

Integrin Activation Enables Sensitive Detection of Functional CD4⁺ and CD8⁺ T Cells: Application to Characterize SARS-CoV-2 Immunity

Anna Schöllhorn, Juliane Schuhmacher, Luciana Besedovsky, Rolf Fendel, Anja T.R. Jensen, Stefan Stevanović, Tanja Lange, Hans Georg Rammensee, Jan Born, Cécile Gouttefangeas^*, Stoyan Dimitrov

^*Corresponding author for this work

8 Citations (Scopus)

Abstract

We have previously shown that conformational change in the β₂-integrin is a very early activation marker that can be detected with fluorescent multimers of its ligand intercellular adhesion molecule (ICAM)-1 for rapid assessment of antigen-specific CD8⁺ T cells. In this study, we describe a modified protocol of this assay for sensitive detection of functional antigen-specific CD4⁺ T cells using a monoclonal antibody (clone m24 Ab) specific for the open, high-affinity conformation of the β₂-integrin. The kinetics of β₂-integrin activation was different on CD4⁺ and CD8⁺ T cells (several hours vs. few minutes, respectively); however, m24 Ab readily stained both cell types 4–6 h after antigen stimulation. With this protocol, we were able to monitor ex vivo effector and memory CD4⁺ and CD8⁺ T cells specific for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), cytomegalovirus (CMV), Epstein–Barr virus (EBV), and hepatitis B virus (HBV) in whole blood or cryopreserved peripheral blood mononuclear cells (PBMCs) of infected or vaccinated individuals. By costaining β₂-integrin with m24 and CD154 Abs, we assessed extremely low frequencies of polyfunctional CD4⁺ T cell responses. The novel assay used in this study allows very sensitive and simultaneous screening of both CD4⁺ and CD8⁺ T cell reactivities, with versatile applicability in clinical and vaccination studies.

Original language	English
Article number	626308
Journal	Frontiers in Immunology
Volume	12
Pages (from-to)	626308
ISSN	1664-3224
DOIs	https://doi.org/10.3389/fimmu.2021.626308
Publication status	Published - 29.03.2021

Funding

We thank S. Heidu for the production of HLA-class I tetramers and excellent technical assistance. The HLA-class II (DRB*11/CMV HPT) multimer was kindly provided by the NIH Tetramer Core Facility. The MR1 tetramer technology was developed jointly by Dr. James McCluskey, Dr. Jamie Rossjohn, and Dr. David Fairlie, and the material was produced by the NIH Tetramer Core Facility as permitted to be distributed by the University of Melbourne. We acknowledge the support of the Open Access Publishing Fund of the University of T?bingen. We have shared a preprint of this manuscript on Research Square [Sch?llhorn et al., (20 )]. Funding. CG, AS, and H-GR were supported by the Deutsche Forschungsgemeinschaft, Collaborative Research Center 1399. CG, SS, and H-GR received support from the Deutsche Forschungsgemeinschaft under Germany's Excellence Strategy?EXC 2180?390900677 [CG, SS, and H-GR sind gef?rdert durch die Deutsche Forschungsgemeinschaft (DFG) im Rahmen der Exzellenzstrategie des Bundes und der L?nder - EXC 2180 ? 390900677]. LB, TL, JB, and SD were supported by grants from the Deutsche Forschungsgemeinschaft (TR-SFB 654, Plasticity and Sleep).

UN SDGs

This output contributes to the following UN Sustainable Development Goals (SDGs)

SDG 3 Good Health and Well-being

Research Areas and Centers

Academic Focus: Center for Brain, Behavior and Metabolism (CBBM)

Coronavirus related work

Research on SARS-CoV-2 / COVID-19

Access to Document

10.3389/fimmu.2021.626308

Cite this

Schöllhorn, A., Schuhmacher, J., Besedovsky, L., Fendel, R., Jensen, A. T. R., Stevanović, S., Lange, T., Rammensee, H. G., Born, J., Gouttefangeas, C., & Dimitrov, S. (2021). Integrin Activation Enables Sensitive Detection of Functional CD4⁺ and CD8⁺ T Cells: Application to Characterize SARS-CoV-2 Immunity. Frontiers in Immunology, 12, 626308. Article 626308. https://doi.org/10.3389/fimmu.2021.626308

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title = "Integrin Activation Enables Sensitive Detection of Functional CD4+ and CD8+ T Cells: Application to Characterize SARS-CoV-2 Immunity",

abstract = "We have previously shown that conformational change in the β2-integrin is a very early activation marker that can be detected with fluorescent multimers of its ligand intercellular adhesion molecule (ICAM)-1 for rapid assessment of antigen-specific CD8+ T cells. In this study, we describe a modified protocol of this assay for sensitive detection of functional antigen-specific CD4+ T cells using a monoclonal antibody (clone m24 Ab) specific for the open, high-affinity conformation of the β2-integrin. The kinetics of β2-integrin activation was different on CD4+ and CD8+ T cells (several hours vs. few minutes, respectively); however, m24 Ab readily stained both cell types 4–6 h after antigen stimulation. With this protocol, we were able to monitor ex vivo effector and memory CD4+ and CD8+ T cells specific for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), cytomegalovirus (CMV), Epstein–Barr virus (EBV), and hepatitis B virus (HBV) in whole blood or cryopreserved peripheral blood mononuclear cells (PBMCs) of infected or vaccinated individuals. By costaining β2-integrin with m24 and CD154 Abs, we assessed extremely low frequencies of polyfunctional CD4+ T cell responses. The novel assay used in this study allows very sensitive and simultaneous screening of both CD4+ and CD8+ T cell reactivities, with versatile applicability in clinical and vaccination studies.",

author = "Anna Sch{\"o}llhorn and Juliane Schuhmacher and Luciana Besedovsky and Rolf Fendel and Jensen, \{Anja T.R.\} and Stefan Stevanovi{\'c} and Tanja Lange and Rammensee, \{Hans Georg\} and Jan Born and C{\'e}cile Gouttefangeas and Stoyan Dimitrov",

note = "Funding Information: We thank S. Heidu for the production of HLA-class I tetramers and excellent technical assistance. The HLA-class II (DRB*11/CMV HPT) multimer was kindly provided by the NIH Tetramer Core Facility. The MR1 tetramer technology was developed jointly by Dr. James McCluskey, Dr. Jamie Rossjohn, and Dr. David Fairlie, and the material was produced by the NIH Tetramer Core Facility as permitted to be distributed by the University of Melbourne. We acknowledge the support of the Open Access Publishing Fund of the University of T?bingen. We have shared a preprint of this manuscript on Research Square [Sch?llhorn et al., (20 )]. Funding. CG, AS, and H-GR were supported by the Deutsche Forschungsgemeinschaft, Collaborative Research Center 1399. CG, SS, and H-GR received support from the Deutsche Forschungsgemeinschaft under Germany's Excellence Strategy?EXC 2180?390900677 [CG, SS, and H-GR sind gef?rdert durch die Deutsche Forschungsgemeinschaft (DFG) im Rahmen der Exzellenzstrategie des Bundes und der L?nder - EXC 2180 ? 390900677]. LB, TL, JB, and SD were supported by grants from the Deutsche Forschungsgemeinschaft (TR-SFB 654, Plasticity and Sleep). Publisher Copyright: {\textcopyright} Copyright {\textcopyright} 2021 Sch{\"o}llhorn, Schuhmacher, Besedovsky, Fendel, Jensen, Stevanovi{\'c}, Lange, Rammensee, Born, Gouttefangeas and Dimitrov. Copyright: Copyright 2021 Elsevier B.V., All rights reserved.",

year = "2021",

month = mar,

day = "29",

doi = "10.3389/fimmu.2021.626308",

language = "English",

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Schöllhorn, A, Schuhmacher, J, Besedovsky, L, Fendel, R, Jensen, ATR, Stevanović, S, Lange, T, Rammensee, HG, Born, J, Gouttefangeas, C & Dimitrov, S 2021, 'Integrin Activation Enables Sensitive Detection of Functional CD4⁺ and CD8⁺ T Cells: Application to Characterize SARS-CoV-2 Immunity', Frontiers in Immunology, vol. 12, 626308, pp. 626308. https://doi.org/10.3389/fimmu.2021.626308

TY - JOUR

T1 - Integrin Activation Enables Sensitive Detection of Functional CD4+ and CD8+ T Cells: Application to Characterize SARS-CoV-2 Immunity

AU - Schöllhorn, Anna

AU - Schuhmacher, Juliane

AU - Besedovsky, Luciana

AU - Fendel, Rolf

AU - Jensen, Anja T.R.

AU - Stevanović, Stefan

AU - Lange, Tanja

AU - Rammensee, Hans Georg

AU - Born, Jan

AU - Gouttefangeas, Cécile

AU - Dimitrov, Stoyan

N1 - Funding Information: We thank S. Heidu for the production of HLA-class I tetramers and excellent technical assistance. The HLA-class II (DRB*11/CMV HPT) multimer was kindly provided by the NIH Tetramer Core Facility. The MR1 tetramer technology was developed jointly by Dr. James McCluskey, Dr. Jamie Rossjohn, and Dr. David Fairlie, and the material was produced by the NIH Tetramer Core Facility as permitted to be distributed by the University of Melbourne. We acknowledge the support of the Open Access Publishing Fund of the University of T?bingen. We have shared a preprint of this manuscript on Research Square [Sch?llhorn et al., (20 )]. Funding. CG, AS, and H-GR were supported by the Deutsche Forschungsgemeinschaft, Collaborative Research Center 1399. CG, SS, and H-GR received support from the Deutsche Forschungsgemeinschaft under Germany's Excellence Strategy?EXC 2180?390900677 [CG, SS, and H-GR sind gef?rdert durch die Deutsche Forschungsgemeinschaft (DFG) im Rahmen der Exzellenzstrategie des Bundes und der L?nder - EXC 2180 ? 390900677]. LB, TL, JB, and SD were supported by grants from the Deutsche Forschungsgemeinschaft (TR-SFB 654, Plasticity and Sleep). Publisher Copyright: © Copyright © 2021 Schöllhorn, Schuhmacher, Besedovsky, Fendel, Jensen, Stevanović, Lange, Rammensee, Born, Gouttefangeas and Dimitrov. Copyright: Copyright 2021 Elsevier B.V., All rights reserved.

PY - 2021/3/29

Y1 - 2021/3/29

N2 - We have previously shown that conformational change in the β2-integrin is a very early activation marker that can be detected with fluorescent multimers of its ligand intercellular adhesion molecule (ICAM)-1 for rapid assessment of antigen-specific CD8+ T cells. In this study, we describe a modified protocol of this assay for sensitive detection of functional antigen-specific CD4+ T cells using a monoclonal antibody (clone m24 Ab) specific for the open, high-affinity conformation of the β2-integrin. The kinetics of β2-integrin activation was different on CD4+ and CD8+ T cells (several hours vs. few minutes, respectively); however, m24 Ab readily stained both cell types 4–6 h after antigen stimulation. With this protocol, we were able to monitor ex vivo effector and memory CD4+ and CD8+ T cells specific for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), cytomegalovirus (CMV), Epstein–Barr virus (EBV), and hepatitis B virus (HBV) in whole blood or cryopreserved peripheral blood mononuclear cells (PBMCs) of infected or vaccinated individuals. By costaining β2-integrin with m24 and CD154 Abs, we assessed extremely low frequencies of polyfunctional CD4+ T cell responses. The novel assay used in this study allows very sensitive and simultaneous screening of both CD4+ and CD8+ T cell reactivities, with versatile applicability in clinical and vaccination studies.

AB - We have previously shown that conformational change in the β2-integrin is a very early activation marker that can be detected with fluorescent multimers of its ligand intercellular adhesion molecule (ICAM)-1 for rapid assessment of antigen-specific CD8+ T cells. In this study, we describe a modified protocol of this assay for sensitive detection of functional antigen-specific CD4+ T cells using a monoclonal antibody (clone m24 Ab) specific for the open, high-affinity conformation of the β2-integrin. The kinetics of β2-integrin activation was different on CD4+ and CD8+ T cells (several hours vs. few minutes, respectively); however, m24 Ab readily stained both cell types 4–6 h after antigen stimulation. With this protocol, we were able to monitor ex vivo effector and memory CD4+ and CD8+ T cells specific for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), cytomegalovirus (CMV), Epstein–Barr virus (EBV), and hepatitis B virus (HBV) in whole blood or cryopreserved peripheral blood mononuclear cells (PBMCs) of infected or vaccinated individuals. By costaining β2-integrin with m24 and CD154 Abs, we assessed extremely low frequencies of polyfunctional CD4+ T cell responses. The novel assay used in this study allows very sensitive and simultaneous screening of both CD4+ and CD8+ T cell reactivities, with versatile applicability in clinical and vaccination studies.

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