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Background: We previously showed that the bacterial lipopeptide Pam 3 Cys-Ser-Ser, meanwhile established as a toll-like receptor (TLR) 1/2 ligand, acts as a strong adjuvant for the induction of virus specific CD8 + T cells in mice, when covalently coupled to a synthetic peptide., Case Presentation: We now designed a new water-soluble synthetic Pam 3 Cys-derivative, named XS15 and characterized it in vitro by a TLR2 NF-κB luciferase reporter assay. Further, the capacity of XS15 to activate immune cells and stimulate peptide-specific CD8 + T and NK cells by 6-sulfo LacNAc + monocytes was assessed by flow cytometry as well as cytokine induction using immunoassays. The induction of a functional immune response after vaccination of a volunteer with viral peptides was assessed by ELISpot assay and flow cytometry in peripheral blood cells and infiltrating cells at the vaccination site, as well as by immunohistochemistry and imaging. XS15 induced strong ex vivo CD8 + and T H 1 CD4 + responses in a human volunteer upon a single injection of XS15 mixed to uncoupled peptides in a water-in-oil emulsion (Montanide™ ISA51 VG). A granuloma formed locally at the injection site containing highly activated functional CD4 + and CD8 + effector memory T cells. The total number of vaccine peptide-specific functional T cells was experimentally assessed and estimated to be 3.0 × 10 5 in the granuloma and 20.5 × 10 6 in peripheral blood., Conclusion: Thus, in one volunteer we show a granuloma forming by peptides combined with an efficient adjuvant in a water-in-oil-emulsion, inducing antigen specific T cells detectable in circulation and at the vaccination site, after one single vaccination only. The ex vivo T cell responses in peripheral blood were detectable for more than one year and could be strongly boosted by a second vaccination. Hence, XS15 is a promising adjuvant candidate for peptide vaccination, in particular for tumor peptide vaccines in a personalized setting.

With the recent results of promising cancer vaccines and immunotherapy1-5, immune monitoring has become increasingly relevant for measuring treatment-induced effects on T cells, and an essential tool for shedding light on the mechanisms responsible for a successful treatment. Flow cytometry is the canonical multi-parameter assay for the fine characterization of single cells in solution, and is ubiquitously used in pre-clinical tumor immunology and in cancer immunotherapy trials. Current state-of-the-art polychromatic flow cytometry involves multi-step, multi-reagent assays followed by sample acquisition on sophisticated instruments capable of capturing up to 20 parameters per cell at a rate of tens of thousands of cells per second. Given the complexity of flow cytometry assays, reproducibility is a major concern, especially for multi-center studies. A promising approach for improving reproducibility is the use of automated analysis borrowing from statistics, machine learning and information visualization21-23, as these methods directly address the subjectivity, operator-dependence, labor-intensive and low fidelity of manual analysis. However, it is quite time-consuming to investigate and test new automated analysis techniques on large data sets without some centralized information management system. For large-scale automated analysis to be practical, the presence of consistent and high-quality data linked to the raw FCS files is indispensable. In particular, the use of machine-readable standard vocabularies to characterize channel metadata is essential when constructing analytic pipelines to avoid errors in processing, analysis and interpretation of results. For automation, this high-quality metadata needs to be programmatically accessible, implying the need for a consistent Application Programming Interface (API). In this manuscript, we propose that upfront time spent normalizing flow cytometry data to conform to carefully designed data models enables automated analysis, potentially saving time in the long run. The ReFlow informatics framework was developed to address these data management challenges.

Ex vivo ELISPOT and multimer staining are well-established tests for the assessment of antigen-specific T cells. Many laboratories are now using a period of in vitro stimulation (IVS) to enhance detection. Here, we report the findings of a multi-centre panel organised by the Association for Cancer Immunotherapy Immunoguiding Program to investigate the impact of IVS protocols on the detection of antigen-specific T cells of varying ex vivo frequency. Five centres performed ELISPOT and multimer staining on centrally prepared PBMCs from 3 donors, both ex vivo and following IVS. A harmonised IVS protocol was designed based on the best-performing protocol(s), which was then evaluated in a second phase on 2 donors by 6 centres. All centres were able to reliably detect antigen-specific T cells of high/intermediate frequency both ex vivo (Phase I) and post-IVS (Phase I and II). The highest frequencies of antigen-specific T cells ex vivo were mirrored in the frequencies following IVS and in the detection rates. However, antigen-specific T cells of a low/undetectable frequency ex vivo were not reproducibly detected post-IVS. Harmonisation of the IVS protocol reduced the inter-laboratory variation observed for ELISPOT and multimer analyses by approximately 20 %. We further demonstrate that results from ELISPOT and multimer staining correlated after (P < 0.0001 and R (2) = 0.5113), but not before IVS. In summary, IVS was shown to be a reproducible method that benefitted from method harmonisation.

The validation of assays that quantify antigen-specific T cell responses is critically dependent on cell samples that contain clearly defined measurable numbers of antigen-specific T cells. An important requirement is that such cell samples are handled and analyzed in a comparable fashion to peripheral blood mononuclear cells (PBMC). We performed a proof-of-principle study to show that retrovirally TCR-transduced T cells spiked at defined numbers in autologous PBMC can be used as standard samples for HLA/peptide multimer staining. NY-ESO-1157-165-specific, TCR-transduced CD8+ T cell batches were successfully generated from PBMC of several HLA-A*0201 healthy donors, purified by magnetic cell sorting on the basis of HLA tetramer (TM) staining and expanded with specific antigen in vitro. When subsequently spiked into autologous PBMC, the detection of these CD3+CD8+TM+ T cells was highly accurate with a mean accuracy of 91.6 %. The standard cells can be preserved for a substantial period of time in liquid nitrogen. Furthermore, TM staining of fresh and cryopreserved standard samples diluted at decreasing concentrations into autologous cryopreserved unspiked PBMC revealed that the spiked CD3+CD8+TM+ T cells could be accurately detected at all dilutions in a linear fashion with a goodness-of-fit of over 0.99 at a frequency of at least 0.02 % among the CD3+CD8+ T cell population. Notably, the CD3+CD8+TM+ cells of the standard samples were located exactly within the gates used to analyze patient samples and displayed a similar scatter pattern. The performance of the cryopreserved standard samples in the hands of 5 external investigators was good with an inter-laboratory variation of 32.9 % and the doubtless identification of one outlier.