An optimized flow cytometry protocol for simultaneous detection of T cell activation induced markers and intracellular cytokines: application to SARS-Cov-2 vaccinated individuals
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Abstract
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. 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, assessing 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 (IFNγ, TNFα 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 poorly 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 helper follicular 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.
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SciScore for 10.1101/2022.04.14.22273819: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Ethics Consent: Specimen collection: Blood samples have been obtained from BNT162b2 fully vaccinated healthy volunteers within several weeks after the second dose (Supplemental Table 1, for Vax146-149, which were most frequently used in this study, the average is 22.75 weeks post-boost) and after informed consent (Supplemental Table 1) Sex as a biological variable not detected. Randomization not detected. Blinding not detected. Power Analysis not detected. Table 2: Resources
Antibodies Sentences Resources Materials: Antibodies for flow cytometry staining (Supplemental Table 2 B), monoclonal antibodies against CD28 and CD49d costimulatory molecules, BD GolgiPlug™ protein transport inhibitor (Brefeldin A, BFA), … SciScore for 10.1101/2022.04.14.22273819: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Ethics Consent: Specimen collection: Blood samples have been obtained from BNT162b2 fully vaccinated healthy volunteers within several weeks after the second dose (Supplemental Table 1, for Vax146-149, which were most frequently used in this study, the average is 22.75 weeks post-boost) and after informed consent (Supplemental Table 1) Sex as a biological variable not detected. Randomization not detected. Blinding not detected. Power Analysis not detected. Table 2: Resources
Antibodies Sentences Resources Materials: Antibodies for flow cytometry staining (Supplemental Table 2 B), monoclonal antibodies against CD28 and CD49d costimulatory molecules, BD GolgiPlug™ protein transport inhibitor (Brefeldin A, BFA), BD Stain Buffer supplemented with BSA, and BD Cytofix/Cytoperm™ solution kit were provided by BD. antibodies against CD28suggested: (Novus Cat# NB100-93558, RRID:AB_1236789)CD49dsuggested: NoneMonoclonal antibodies anti-CD28 and /or anti-CD49d at 1 μg/ml were added as co-stimulus. anti-CD28suggested: NoneSoftware and Algorithms Sentences Resources Cell staining: After incubation with BD GolgiPlug™, cells were washed with 100 ul of Stain Buffer to remove culture medium. BD GolgiPlug™suggested: NoneSamples were finally washed with Stain Buffer and re-suspended in 400 μl of PBS in 5 ml polystyrene tubes for acquisition with BD FACSCelesta™ flow cytometer equipped with Blue, Violet and Red lasers. BD FACSCelesta™suggested: NoneFlow cytometry and data analysis: Cells were acquired on a BD FACSCelesta™ flow cytometer and analyzed using BD FACSDiva™ software (v9.0). BD FACSDiva™suggested: (BD FACSDiva Software, RRID:SCR_001456)Results from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).
Results from LimitationRecognizer: We detected the following sentences addressing limitations in the study:The main limitations of the current study are: 1) the experimental model system is rooted in the context of SARS-Cov-2 and 2) the small number of donors and variability. On the first, the study is conducted using vaccinated donors assessed several weeks after BNT62b2 vaccination (Supplemental Table 1) and stimulated with Spike peptides, as well as using control stimulation with PHA, but has not been studied for other T-cell activation conditions. On the second, the study used four vaccinated donors with samples split into several cryopreserved aliquots (106 PBMCs/sample) for comparison across multiple conditions and variability ensued for at least two reasons, i) relatively few activated T cells were detected in each sample, perhaps due to the starting cell numbers and since on average 22.75 weeks had ensued since the last boost (for Vax 146-149), such that in most cases another boost would now be recommended, and ii) the introduction of more random noise by needing to subtract background activation from stimulated activation to properly determine the percentage of AIM+ T cells. Recommended improvements for evaluating individual samples in detail are to consider running multiple replicates, multiple timepoints, evaluation closer to time of boost, and increasing the number of processed cells. Nevertheless, taken altogether the data presented here clearly show the optimal performance of intracellular CD137 and CD69 together with intracellular cytokine detection. In conclusion, ...
Results from TrialIdentifier: No clinical trial numbers were referenced.
Results from Barzooka: We did not find any issues relating to the usage of bar graphs.
Results from JetFighter: We did not find any issues relating to colormaps.
Results from rtransparent:- Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
- Thank you for including a funding statement. Authors are encouraged to include this statement when submitting to a journal.
- No protocol registration statement was detected.
Results from scite Reference Check: We found no unreliable references.
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