Dual-modality imaging of immunofluorescence and imaging mass cytometry for whole-slide imaging and accurate segmentation.

Imaging mass cytometry (IMC) is a powerful technique capable of detecting over 30 markers on a single slide. It has been increasingly used for single-cell-based spatial phenotyping in a wide range of samples. However, it only acquires a rectangle field of view (FOV) with a relatively small size and low image resolution, which hinders downstream analysis. Here, we reported a highly practical dual-modality imaging method that combines high-resolution immunofluorescence (IF) and high-dimensional IMC on the same tissue slide. Our computational pipeline uses the whole-slide image (WSI) of IF as a spatial reference and integrates small-FOV IMC into a WSI of IMC. The high-resolution IF images enable accurate single-cell segmentation to extract robust high-dimensional IMC features for downstream analysis. We applied this method in esophageal adenocarcinoma of different stages, identified the single-cell pathology landscape via reconstruction of WSI IMC images, and demonstrated the advantage of the dual-modality imaging strategy.

Dual-modality imaging of immunofluorescence and imaging mass cytometry for whole slide imaging with accurate single-cell segmentation.

Imaging mass cytometry (IMC) is a powerful multiplexed tissue imaging technology that allows simultaneous detection of more than 30 makers on a single slide. It has been increasingly used for singlecell-based spatial phenotyping in a wide range of samples. However, it only acquires a small, rectangle field of view (FOV) with a low image resolution that hinders downstream analysis. Here, we reported a highly practical dual-modality imaging method that combines high-resolution immunofluorescence (IF) and high-dimensional IMC on the same tissue slide. Our computational pipeline uses the whole slide image (WSI) of IF as a spatial reference and integrates small FOVs IMC into a WSI of IMC. The high-resolution IF images enable accurate single-cell segmentation to extract robust high-dimensional IMC features for downstream analysis. We applied this method in esophageal adenocarcinoma of different stages, identified the single-cell pathology landscape via reconstruction of WSI IMC images, and demonstrated the advantage of the dual-modality imaging strategy. Motivation: Highly multiplexed tissue imaging allows visualization of the spatially resolved expression of multiple proteins at the single-cell level. Although imaging mass cytometry (IMC) using metal isotope-conjugated antibodies has a significant advantage of low background signal and absence of autofluorescence or batch effect, it has a low resolution that hampers accurate cell segmentation and results in inaccurate feature extraction. In addition, IMC only acquires mm 2 -sized rectangle regions, which limits its application and efficiency when studying larger clinical samples with non-rectangle shapes. To maximize the research output of IMC, we developed the dual-modality imaging method based on a highly practical and technical improvement requiring no extra specialized equipment or agents and proposed a comprehensive computational pipeline that combines IF and IMC. The proposed method greatly improves the accuracy of cell segmentation and downstream analysis and is able to obtain whole slide image IMC to capture the comprehensive cellular landscape of large tissue sections.

Compartmentalization and persistence of dominant (regulatory) T cell clones indicates antigen skewing in juvenile idiopathic arthritis.

Autoimmune inflammation is characterized by tissue infiltration and expansion of antigen-specific T cells. Although this inflammation is often limited to specific target tissues, it remains yet to be explored whether distinct affected sites are infiltrated with the same, persistent T cell clones. Here, we performed CyTOF analysis and T cell receptor (TCR) sequencing to study immune cell composition and (hyper-)expansion of circulating and joint-derived Tregs and non-Tregs in juvenile idiopathic arthritis (JIA). We studied different joints affected at the same time, as well as over the course of relapsing-remitting disease. We found that the composition and functional characteristics of immune infiltrates are strikingly similar between joints within one patient, and observed a strong overlap between dominant T cell clones, especially Treg, of which some could also be detected in circulation and persisted over the course of relapsing-remitting disease. Moreover, these T cell clones were characterized by a high degree of sequence similarity, indicating the presence of TCR clusters responding to the same antigens. These data suggest that in localized autoimmune disease, there is autoantigen-driven expansion of both Teffector and Treg clones that are highly persistent and are (re)circulating. These dominant clones might represent interesting therapeutic targets.

Microbial exposure during early human development primes fetal immune cells.

The human fetal immune system begins to develop early during gestation; however, factors responsible for fetal immune-priming remain elusive. We explored potential exposure to microbial agents in utero and their contribution toward activation of memory T cells in fetal tissues. We profiled microbes across fetal organs using 16S rRNA gene sequencing and detected low but consistent microbial signal in fetal gut, skin, placenta, and lungs in the 2nd trimester of gestation. We identified several live bacterial strains including Staphylococcus and Lactobacillus in fetal tissues, which induced in vitro activation of memory T cells in fetal mesenteric lymph node, supporting the role of microbial exposure in fetal immune-priming. Finally, using SEM and RNA-ISH, we visualized discrete localization of bacteria-like structures and eubacterial-RNA within 14th weeks fetal gut lumen. These findings indicate selective presence of live microbes in fetal organs during the 2nd trimester of gestation and have broader implications toward the establishment of immune competency and priming before birth.

Immunome perturbation is present in patients with juvenile idiopathic arthritis who are in remission and will relapse upon anti-TNFα withdrawal.

OBJECTIVES: Biologics treatment with antitumour necrosis factor alpha (TNFα) is efficacious in patients with juvenile idiopathic arthritis (JIA). Despite displaying clinical inactivity during treatment, many patients will flare on cessation of therapy. The inability to definitively discriminate patients who will relapse or continue to remain in remission after therapy withdrawal is currently a major unmet medical need. CD4 T cells have been implicated in active disease, yet how they contribute to disease persistence despite treatment is unknown. METHODS: We interrogated the circulatory reservoir of CD4+ immune subsets at the single-cell resolution with mass cytometry (cytometry by time of flight) of patients with JIA (n=20) who displayed continuous clinical inactivity for at least 6 months with anti-TNFα and were subsequently withdrawn from therapy for 8 months, and scored as relapse or remission. These patients were examined prior to therapy withdrawal for putative subsets that could discriminate relapse from remission. We verified on a separate JIA cohort (n=16) the dysregulation of these circulatory subsets 8 months into therapy withdrawal. The immunological transcriptomic signature of CD4 memory in relapse/remission patients was examined with NanoString. RESULTS: An inflammatory memory subset of CD3+CD4+CD45RA-TNFα+ T cells deficient in immune checkpoints (PD1-CD152-) was present in relapse patients prior to therapy withdrawal. Transcriptomic profiling reveals divergence between relapse and remission patients in disease-centric pathways involving (1) T-cell receptor activation, (2) apoptosis, (3) TNFα, (4) nuclear factor-kappa B and (5) mitogen-activated protein kinase signalling. CONCLUSIONS: A unique discriminatory immunomic and transcriptomic signature is associated with relapse patients and may explain how relapse occurs.