Refined analytical pipeline for the pharmacodynamic assessment of T-cell responses to vaccine antigens

Abstract

Pharmacodynamic assessment of T-cell-based cancer immunotherapies often focus on detecting rare circulating T-cell populations. The therapy-induced immune cells in blood-derived clinical samples are often present in very low frequencies and with the currently available T-cell analytical assays, amplification of the cells of interest prior to analysis is often required. Current approaches aiming to enrich antigen-specific T cells from human Peripheral Blood Mononuclear Cells (PBMCs) depend on in vitro culturing in presence of their cognate peptides and cytokines. In the present work, we improved a standard, publicly available protocol for T-cell immune analyses based on the in vitro expansion of T cells. We used PBMCs from healthy subjects and well-described viral antigens as a model system for optimizing the experimental procedures and conditions. Using the standard protocol, we first demonstrated significant enrichment of antigen-specific T cells, even when their starting frequency ex vivo was low. Importantly, this amplification occurred with high specificity, with no or neglectable enrichment of irrelevant T-cell clones being observed in the cultures. Testing of modified culturing timelines suggested that the protocol can be adjusted accordingly to allow for greater cell yield with strong preservation of the functionality of antigen-specific T cells. Overall, our work has led to the refinement of a standard protocol for in vitro stimulation of antigen-specific T cells and highlighted its reliability and reproducibility. We envision that the optimized protocol could be applied for longitudinal monitoring of rare blood-circulating T cells in scenarios with limited sample material.

Keywords: T-cell expansion; T-cell response; antigen specificity; cell culture; immune monitoring; in vitro stimulation; pipeline; vaccine antigen.

Figure 1

In vitro stimulation (IVS) of human PBMCs with peptides following a publicly available protocol leads to strong enrichment of antigen-specific T cells. (A) Workflow of the used IVS protocol. Figure created with BioRender. (B) % IFNγ+ & TNFα+ within CD4+ or CD8+ T cells upon stimulation of PBMCs from HD005 with peptides #15_long or #15_short either directly ex vivo or after IVS with peptide #15_long (n = 4 IVS cultures). Ex vivo data come from independent experiments. Each culture was tested in the indicated conditions in a single measurement. One culture was not analyzed for recognition of peptide #15_short. Mean ± SD. A multiple t-test approach was used to test for statistical significance (outlined in “Materials and Methods”) (C) % IFNγ+ & TNFα+ within CD4+ or CD8+ T cells upon stimulation of PBMCs from HD011 with peptide #10_short either directly ex vivo or after IVS with peptide #10_long (n = 1 IVS culture). Ex vivo data come from independent experiments. Mean ± SD. (D) % IFNγ+ & TNFα+ within CD4+ or CD8+ T cells upon stimulation of PBMCs from HD011 with peptides #10_long and #10_short after IVS with either of peptides #10_long and #10_short (n = 1 IVS culture per peptide). (E) BV650 fluorophore-labelled tetramers consisting of HLA-A11:01 alleles loaded with peptide #10_short were used to stain HD011 PBMCs either directly ex vivo or after IVS with peptide #10_short. (F) % IFNγ+ & TNFα+ within CD4+ or CD8+ T cells upon stimulation of PBMCs from HD008 with four viral peptides (single and pooled) either directly ex vivo or after IVS with the pool of the four viral peptides. (G) Fluorophore-labelled tetramers consisting of HLA-A2:01 alleles loaded with minimal epitopes deriving from the melanoma antigens MART-1, NY-ESO-1, MAGE-A3 and gp100 were used to stain PBMCs from MM011234 melanoma patient either directly ex vivo or after IVS with the peptide pool (n = 1 IVS culture). As negative control, a non-immunogenic HLA-A2:01 ligand was used.

Figure 2

IVS of healthy donor PBMCs specifically enriches T cells reactive to the IVS peptide. (A) % IFNγ+ & TNFα+ within CD4+ or CD8+ T cells upon LAC stimulation of PBMCs from the three healthy donors either directly ex vivo or after IVS with the viral peptides presented in Figure 1 . (B) % IFNγ+ & TNFα+ within CD8+ T cells upon stimulation of PBMCs from HD005 with peptides #16_long, #16_short or their pool either directly ex vivo or after IVS with peptide #15_long (n = 3 IVS cultures). Ex vivo data come from one experiment. Each culture was tested in the indicated conditions in a single measurement. Mean ± SD. (C) % IFNγ+ & TNFα+ within CD8+ T cells upon stimulation of PBMCs from HD008 with a pool of the short peptides #1, #2 and #4 after IVS with peptide #5_short. (n = 2 IVS cultures). Each culture was tested in the indicated conditions in a single measurement. Mean ± SD. (D) Well images from IFNγ ELISpot assay after stimulation of #10_short-expanded HD011 PBMCs with peptides #10_short or #5_short. Top-left numbers indicate the counted Spot Forming Units (SFUs) per 150.000 cells. (E) BV605 fluorophore-labelled tetramers consisting of HLA-A02:01 alleles loaded with peptide #5_short were used to stain HD011 PBMCs either directly ex vivo or after IVS with peptide #10_short (n = 1 IVS culture). (F) % IFNγ+ & TNFα+ within CD4+ or CD8+ T cells upon stimulation of PBMCs from HD011 with a pool of five irrelevant murine peptides or peptide #10_short after IVS with the pool of the murine peptides (n = 1 IVS culture). Murine peptide pool stimulation was tested in technical duplicates. Mean ± SD. (G) BV650 fluorophore-labelled tetramers consisting of HLA-A11:01 alleles loaded with peptide #10_short were used to stain HD011 PBMCs after IVS with a pool of five murine peptides (n = 1 IVS culture). (H) Well images from IFNγ ELISpot assay and (I) extrapolated SFUs per 1 x 10⁶ cells after overnight stimulation of HD005 PBMCs with donor-irrelevant, murine peptides and CEFT-I pool either directly ex vivo or after IVS with this pool (n = 1 IVS culture). Top-left numbers in (H) indicate the counted SFUs per 500.000 cells (ex vivo) or 300.000 cells (IVS). IVS cells was analyzed in two separate experiments. Mean ± SD.

Figure 3

The publicly available IVS protocol can be adjusted to increase the fraction of functional, antigen-specific T cells and the cell yield of the cultures. (A) Illustration of the different IVS culturing schemes tested. Figure created with BioRender. (B) % IFNγ+ & TNFα+ within CD8+ T cells upon stimulation of HD005 PBMCs with peptide #15_short following IVS with peptide #15_long using the alternative culturing scheme (n = 3 IVS cultures). Each culture was tested in the indicated conditions in a single measurement. Mean ± SD. (C) % IFNγ+ & TNFα+ within CD8+ T cells upon stimulation of HD008 PBMCs with peptide #5_short following IVS with peptide #5_short using the standard or the alternative culturing scheme (n = 2 IVS cultures). Each culture was tested in the indicated conditions in a single measurement. Mean ± SD. (D) % IFNγ+ & TNFα+ within CD8+ T cells upon stimulation of HD005 PBMCs with peptide #15_short following IVS with peptide #15_long using the extended version of the standard IVS culturing timeline (n = 3 IVS cultures). Mean ± SD. (E) % IFNγ+ & TNFα+ within CD8+ T cells upon stimulation of HD008 PBMCs with peptide #5_short following IVS with peptide #5_short using the extended version of the standard IVS culturing timeline (n = 2 IVS cultures). Mean ± SD. (F) % IFNγ+ & TNFα+ within CD8+ T cells upon stimulation of HD005 PBMCs with peptide #15_short following IVS with peptide #15_long using the extended version of the alternative IVS culturing timeline (n = 3 IVS cultures). Mean ± SD. (G) Fold-increase of the PBMC input numbers after IVS of PBMCs from HD005 and HD008 following the different IVS culturing schemes presented in Figure 3A . For the calculation of the fold-increase for standard HD005, the IVS cultures from Figure 1B were used. (H) % viability of harvested cells in the HD005 IVS cultures (n = 3 IVS cultures per expansion protocol) and HD008 IVS cultures (n = 2 IVS cultures per expansion protocol). Mean ± SD.