Harmonization and qualification of intracellular cytokine staining to measure influenza-specific CD4+ T cell immunity within the FLUCOP consortium

Abstract

Despite the knowledge that cell-mediated immunity (CMI) contributes to the reduction of severe influenza infection, transmission, and disease outcome, the correlates of protection for cell-mediated immunity remain still unclear. Therefore, measuring the magnitude and quality of influenza-specific T cell responses in a harmonized way is of utmost importance to improve characterisation of vaccine-induced immunity across different clinical trials. The present study, conducted as part of the FLUCOP project, describes the development of a consensus protocol for the intracellular cytokine staining (ICS) assay, in order to reduce inter-laboratory variability, and its qualification. In order to develop a consensus protocol, the study was divided into different stages. Firstly, two pilot studies evaluated critical parameters in the analytical (read-outs) and post-analytical (gating strategies and data analysis) methods applied by eight different laboratories within the FLUCOP consortium. The methods were then harmonized by fixing the critical parameters and the subsequent consensus protocol was then qualified by one FLUCOP member. The antigen-specific cell population was defined as polypositive CD4+ T cells (i.e. positive for at least two markers among CD40L/IFNγ/IL2/TNFα), which was shown to be the most sensitive and specific read-out. The qualification of this consensus protocol showed that the quantification of polypositive CD4+ T cells was precise, linear and accurate, and sensitive with a lower limit of quantification of 0.0335% antigen-specific polypositive CD4+ T cells. In conclusion, we provide the description of a harmonized ICS assay, which permits quantitative and qualitative evaluation of influenza vaccine-induced T cell responses. Application of this harmonized assay may allow for future comparisons of T cell responses to different influenza vaccines. It may facilitate future assessments of potential correlates of protection with the promise of application across other pathogens.

Immune responses in healthy adults elicited by a bivalent norovirus vaccine candidate composed of GI.4 and GII.4 VLPs without adjuvant

“…CD8 + T cell responses were below the lower limit of quantification set at 0.0335% CD8 + polypositive cells ( 26 ) and therefore interpreted as very low to negative. This was not surprising as the PBMC had been stimulated overnight with boiled VLPs and, e.g., not with peptide libraries representing the VP1 proteins of GI.4 Chiba 407 (1987) and GII.4 Aomori 2 (2006).…”

“…4 Chiba 407 [1987] + GII. 4 Aomori 2 [2006]) vaccine consisting of VLP at two dose levels (50 µg or 150 µg each) manufactured using the magnICON ® plant-based, transient expression system (23,24). Safety and initial evaluation of immune responses for the 60 healthy adult participants of the trial were already published in Leroux-Roels et al (22).…”

T cells, more than antibodies, may prevent symptoms developing from respiratory syncytial virus infections in older adults

“…CD4+ and CD8+ T-cell frequencies were measured by the in vitro stimulation of PBMC cultures and staining for immune markers ( 15 ). Briefly, PBMCs were stimulated with (i) RSV-A lysate, (ii) RSV-B lysate; or pools of peptides (15mers overlapping by 11) spanning the coding sequences for (iii) RSV-A F, (iv) M2-1, or (v) N; or PBMCs were not stimulated (cultured with medium only) for background measurements.…”

SARS-CoV-2 epitope-specific T cells: Immunity response feature, TCR repertoire characteristics and cross-reactivity

“…With the advancement of immunology-related basic research, the detection technologies for antigen-specific T lymphocytes are also rapidly developing and improving. ELISpot and ICS are classical techniques widely used to detect and enumerate activated antigen-specific T cells secreting cytokines such as IFN-g or TNF-a (63,64). Both assays combined with SARS-CoV-2 overlapping peptide pools or single epitope stimulation of peripheral blood mononuclear cells (PBMCs) or tissue samples are widely used in the detecting and identifying SARS-CoV-2 specific T cells, with the advantage of high sensitivity (17,22,28,65,66).…”