PMID- 36842524
OWN - NLM
STAT- MEDLINE
DCOM- 20230403
LR  - 20230622
IS  - 1872-7905 (Electronic)
IS  - 0022-1759 (Print)
IS  - 0022-1759 (Linking)
VI  - 515
DP  - 2023 Apr
TI  - An optimized flow cytometry protocol for simultaneous detection of T cell 
      activation induced markers and intracellular cytokines: Application to SARS-CoV-2 
      immune individuals.
PG  - 113443
LID - S0022-1759(23)00025-X [pii]
LID - 10.1016/j.jim.2023.113443 [doi]
AB  - Antigen (ag)-specific T cell analysis is an important step for investigation of 
      cellular immunity in many settings, such as infectious diseases, cancer and 
      vaccines. Multiparameter flow cytometry has advantages in studying both the 
      rarity and heterogeneity of these cells. In the cellular immunologist's toolbox, 
      the expression of activation-induced markers (AIM) following antigen exposure has 
      made possible the study and sorting of ag-specific T cells without using human 
      leukocyte antigen (HLA)-multimers. In parallel, assessing the cytokine profile of 
      responding T cells would support a more comprehensive description of the ongoing 
      immune response by providing information related to cell function, such as 
      polarization and effector activity. Here, a method and flow cytometry panel were 
      optimized to combine the detection of activated CD4+ and CD8+ T cells in a 
      TCR-dependent manner with the evaluation of cytokine production by intracellular 
      staining, without affecting the positivity of activation markers. In particular, 
      the expression of CD134 (OX40) and CD69 have been tested in conjunction with 
      intracellular (ic) CD137 (4-1BB) to detect SARS-CoV-2 Spike protein-specific 
      activated T cells. In our setting, CD134 provided minimal contribution to detect 
      the pool of AIM+ T cells, whereas a key role was described for ic-CD69 which was 
      co-expressed with ic-CD137 in both CD4+ and CD8+ lymphocytes. Moreover, the 
      analysis of TCR-triggered cytokine-producing T cells (IFNgamma, TNFalpha and IL-2 were 
      assessed) further confirmed the capacity of ic-CD69 to identify functionally 
      responsive antigen-specific T cells which were often largely negative or weakly 
      positive for CD134 expression. In parallel, the use of CD45RA, CCR7 and CXCR5 
      allowed us to describe the T cell matuarion curve and detect T follicular helper 
      (Tfh) CD4+ cells, including the antigen specific activated subsets. In 
      conclusion, we optimized a method and flow cytometry panel combining assessment 
      of activation induced markers and intracellular cytokines that will be useful for 
      measuring TCR stimulation-dependent activation of CD4+ and CD8+ T cells.
CI  - Copyright (c) 2023 The Authors. Published by Elsevier B.V. All rights reserved.
FAU - Altosole, Tiziana
AU  - Altosole T
AD  - IRCCS Ospedale Policlinico San Martino, Genoa, Italy.
FAU - Rotta, Gianluca
AU  - Rotta G
AD  - BD Biosciences, Europe.
FAU - Uras, Chiara R M
AU  - Uras CRM
AD  - Department of Experimental Medicine, Centre of Excellence for Biomedical 
      Research, University of Genoa, Italy.
FAU - Bornheimer, Scott J
AU  - Bornheimer SJ
AD  - BD Biosciences, USA.
FAU - Fenoglio, Daniela
AU  - Fenoglio D
AD  - Department of Internal Medicine, University of Genoa, Italy; Biotherapy Unit, 
      IRCCS Ospedale Policlinico San Martino, Genoa, Italy. Electronic address: 
      daniela.fenoglio@unige.it.
LA  - eng
PT  - Journal Article
DEP - 20230224
PL  - Netherlands
TA  - J Immunol Methods
JT  - Journal of immunological methods
JID - 1305440
RN  - 0 (Cytokines)
RN  - 0 (spike protein, SARS-CoV-2)
RN  - 0 (Antigens)
RN  - 0 (Receptors, Antigen, T-Cell)
SB  - IM
MH  - Humans
MH  - *Cytokines/metabolism
MH  - Flow Cytometry
MH  - SARS-CoV-2/metabolism
MH  - Lymphocyte Activation
MH  - *COVID-19/diagnosis
MH  - CD8-Positive T-Lymphocytes
MH  - Antigens
MH  - Receptors, Antigen, T-Cell
MH  - CD4-Positive T-Lymphocytes
PMC - PMC9957341
OTO - NOTNLM
OT  - Flow cytometry
OT  - SARS-Cov-2 vaccine
OT  - T cell mediated immunity
COIS- Declaration of Competing Interest Gianluca Rotta and Scott J. Bornheimer are 
      employees of Becton, Dickinson and Company.
EDAT- 2023/02/27 06:00
MHDA- 2023/04/03 06:41
PMCR- 2023/02/24
CRDT- 2023/02/26 19:26
PHST- 2022/08/01 00:00 [received]
PHST- 2023/02/16 00:00 [revised]
PHST- 2023/02/16 00:00 [accepted]
PHST- 2023/04/03 06:41 [medline]
PHST- 2023/02/27 06:00 [pubmed]
PHST- 2023/02/26 19:26 [entrez]
PHST- 2023/02/24 00:00 [pmc-release]
AID - S0022-1759(23)00025-X [pii]
AID - 113443 [pii]
AID - 10.1016/j.jim.2023.113443 [doi]
PST - ppublish
SO  - J Immunol Methods. 2023 Apr;515:113443. doi: 10.1016/j.jim.2023.113443. Epub 2023 
      Feb 24.