Abstract Dendritic cells (DCs) are short-lived immune cells that continuously roam our body in search for foreign or self-antigens. Upon acquisition of antigen, they mature and start migrating to the lymph node to present the antigen to naïve T cells. Depending on the context wherein the antigen is acquired, DCs will mature in a homeostatic or immunogenic manner. So far, the field is lacking proper tools to distinguish between the two maturation states. Most maturation markers are shared between the two states and therefore inappropriate to use. Still, defining the proper maturation type is crucial as it determines how the DCs will instruct the T cells towards antigen expressing cells. In this study, we used a lipid nanoparticle (LNP)-based approach to steer DC maturation pathways in vivo. CITE-seq analysis allowed us to design a panel of flow cytometry markers that reliably annotates the two DC maturation states, as validated in an infection and in a tumor model. Furthermore, the data corroborated that uptake of empty LNPs in DCs induces their homeostatic maturation, in contrast to uptake of mRNA-LNPs or TLR ligand-adjuvanted LNPs, leading to distinct effector T cell outputs. This reveals that LNPs themselves are not being decoded as “danger” by cDC1s, and that the cargo is essential to provide adjuvants activity, which is highly relevant for targeted design of LNP-based therapies.

Abstract The continuous engulfment of apoptotic cells initiates a homeostatic maturation program in conventional type I dendritic cells (cDC1s), hallmarked by the activation of the transcription factor LXRb, which mediates cholesterol efflux and dampens interferon stimulated gene expression. cDC1s are characterized by a high basal activation of the unfolded protein response (UPR) sensor IRE1, without concomitant induction of a proper UPR gene signature, a finding that has puzzled the field. Here we show that in absence of IRE1, the homeostatic maturation of cDC1s is blocked, while homeostatic maturation of cDC2s remains unaffected. IRE1 activation is strictly dependent on apoptotic cell engulfment and cholesterol influx, explaining its cDC1 subset specific activity. Stimulation of IRE1 endonuclease activity in cDC1s leads to a Regulated IRE1 Dependent Decay (RIDD) response, targeting miRNAs rather than mRNAs. This causes the degradation of miRNA-92a, which targets the cholesterol efflux transporter Abcg1. Loss of IRE1 leads to defects in cholesterol efflux in mature cDC1s and concomitant cell death, while cDC2s do not show any defects. Blocking miRNA synthesis or enforcing cholesterol efflux by treatment with reconstituted high-density lipoproteins rescues cDC1s from cell death. These data highlight the central role of IRE1 as a sensor of cholesterol influx in the ER, extending IRE1’s function beyond its canonical role in protein folding. Furthermore, they underscore the tight control of cholesterol metabolism during cDC1 maturation, uncovering a second pathway to coordinate cholesterol efflux that acts in parallel to LXRb.

Over the past decade, the flow cytometry field has witnessed significant advancements in the number of fluorochromes that can be detected. This enables researchers to analyze more than 40 markers simultaneously on thousands of cells per second. However, with this increased complexity and multiplicity of markers, the manual dispensing of antibodies for flow cytometry experiments has become laborious, time-consuming, and prone to errors. An automated antibody dispensing system could provide a potential solution by enhancing the efficiency, and by improving data quality by faithfully dispensing the fluorochrome-conjugated antibodies and by enabling the easy addition of extra controls. In this study, a comprehensive comparison of different liquid handlers for dispensing fluorochrome-labeled antibodies was conducted for the preparation of flow cytometry stainings. The evaluation focused on key criteria including dispensing time, dead volume, and reliability of dispensing. After benchmarking, the I.DOT, a non-contact liquid handler, was selected and optimized in more detail. In the end, the I.DOT was able to prepare a 25-marker panel in 20 min, including the full stain, all FMOs and all single stain controls for cells and beads. Having all these controls improved the validation of the panel, visualization, and analysis of the data. Thus, automated antibody dispensing by dispensers such as the I.DOT reduces time and errors, enhances data quality, and can be easily integrated in an automated workflow to prepare samples for flow cytometry.
© 2024 The Authors. Cytometry Part A published by Wiley Periodicals LLC on behalf of International Society for Advancement of Cytometry.

We have recently shown extensive sequence and conformational homology between tumor-associated antigens (TAAs) and antigens derived from microorganisms (MoAs). The present study aimed to assess the breadth of T-cell recognition specific to MoAs and the corresponding TAAs in healthy subjects (HS) and patients with cancer (CP).
A library of > 100 peptide-MHC (pMHC) combinations was used to generate DNA-barcode labelled multimers. Homologous peptides were selected from the Cancer Antigenic Peptide Database, as well as Bacteroidetes/Firmicutes-derived peptides. They were incubated with CD8 + T cells from the peripheral blood of HLA-A*02:01 healthy individuals (n = 10) and cancer patients (n = 16). T cell recognition was identified using tetramer-staining analysis. Cytotoxicity assay was performed using as target cells TAP-deficient T2 cells loaded with MoA or the paired TuA.
A total of 66 unique pMHC recognized by CD8+ T cells across all groups were identified. Of these, 21 epitopes from microbiota were identified as novel immunological targets. Reactivity against selected TAAs was observed for both HS and CP. pMHC tetramer staining confirmed CD8+ T cell populations cross-reacting with CTA SSX2 and paired microbiota epitopes. Moreover, PBMCs activated with the MoA where shown to release IFNγ as well as to exert cytotoxic activity against cells presenting the paired TuA.
Several predicted microbiota-derived MoAs are recognized by T cells in HS and CP. Reactivity against TAAs was observed also in HS, primed by the homologous bacterial antigens. CD8+ T cells cross-reacting with MAGE-A1 and paired microbiota epitopes were identified in three subjects. Therefore, the microbiota can elicit an extensive repertoire of natural memory T cells to TAAs, possibly able to control tumor growth ("natural anti-cancer vaccination"). In addition, non-self MoAs can be included in preventive/therapeutic off-the-shelf cancer vaccines with more potent anti-tumor efficacy than those based on TAAs.
© 2024. The Author(s).

Peptide-loaded MHC class I (pMHC-I) multimers have revolutionized our capabilities to monitor disease-associated T cell responses with high sensitivity and specificity. To improve the discovery of T cell receptors (TCR) targeting neoantigens of individual tumor patients with recombinant MHC molecules, we developed a peptide-loadable MHC class I platform termed MediMer. MediMers are based on soluble disulfide-stabilized β2-microglobulin/heavy chain ectodomain single-chain dimers (dsSCD) that can be easily produced in large quantities in eukaryotic cells and tailored to individual patients' HLA allotypes with only little hands-on time. Upon transient expression in CHO-S cells together with ER-targeted BirA biotin ligase, biotinylated dsSCD are purified from the cell supernatant and are ready to use. We show that CHO-produced dsSCD are free of endogenous peptide ligands. Empty dsSCD from more than 30 different HLA-A,B,C allotypes, that were produced and validated so far, can be loaded with synthetic peptides matching the known binding criteria of the respective allotypes, and stored at low temperature without loss of binding activity. We demonstrate the usability of peptide-loaded dsSCD multimers for the detection of human antigen-specific T cells with comparable sensitivities as multimers generated with peptide-tethered β2m-HLA heavy chain single-chain trimers (SCT) and wild-type peptide-MHC-I complexes prior formed in small-scale refolding reactions. Using allotype-specific, fluorophore-labeled competitor peptides, we present a novel dsSCD-based peptide binding assay capable of interrogating large libraries of in silico predicted neoepitope peptides by flow cytometry in a high-throughput and rapid format. We discovered rare T cell populations with specificity for tumor neoepitopes and epitopes from shared tumor-associated antigens in peripheral blood of a melanoma patient including a so far unreported HLA-C*08:02-restricted NY-ESO-1-specific CD8+ T cell population. Two representative TCR of this T cell population, which could be of potential value for a broader spectrum of patients, were identified by dsSCD-guided single-cell sequencing and were validated by cognate pMHC-I multimer staining and functional responses to autologous peptide-pulsed antigen presenting cells. By deploying the technically accessible dsSCD MHC-I MediMer platform, we hope to significantly improve success rates for the discovery of personalized neoepitope-specific TCR in the future by being able to also cover rare HLA allotypes.
Copyright © 2024 Meyer, Parpoulas, Barthélémy, Becker, Charoentong, Lyu, Börsig, Bulbuc, Tessmer, Weinacht, Ibberson, Schmidt, Pipkorn, Eichmüller, Steinberger, Lindner, Poschke, Platten, Fröhling, Riemer, Hassel, Roberti, Jäger, Zörnig and Momburg.