SingletSeeker: an unsupervised clustering approach for automated singlet discrimination in cytometry.

Flow cytometry is a high-throughput, high-dimensional technique that generates large sets of single-cell data. Prior to analyzing this data, it is common to exclude any events that contain two or more cells, multiplets, to ensure downstream analysis and quantification is of single-cell events, singlets, only. The process of singlet discrimination is critical yet fundamentally subjective and time-consuming; it is performed manually by the user, where the proper exclusion of multiplets depends on the user's expertise and often varies from experiment to experiment.

OMIP-102: 50-color phenotyping of the human immune system with in-depth assessment of T cells and dendritic cells.

We report the development of an optimized 50-color spectral flow cytometry panel designed for the in-depth analysis of the immune system in human blood and tissues, with the goal of maximizing the amount of information that can be collected using currently available flow cytometry platforms. We established and tested this panel using peripheral blood mononuclear cells (PBMCs), but included CD45 to enable its future use for the analysis of human tissue samples. The panel contains lineage markers for all major immune cell subsets, and an extensive set of phenotyping markers focused on the activation and differentiation status of the T cell and dendritic cell (DC) compartment.

Signals that control MAIT cell function in healthy and inflamed human tissues.

Mucosal-associated invariant T (MAIT) cells have a semi-invariant T-cell receptor that allows recognition of antigen in the context of the MHC class I-related (MR1) protein. Metabolic intermediates of the riboflavin synthesis pathway have been identified as MR1-restricted antigens with agonist properties. As riboflavin synthesis occurs in many bacterial species, but not human cells, it has been proposed that the main purpose of MAIT cells is antibacterial surveillance and protection.

50-color phenotyping of the human immune system with in-depth assessment of T cells and dendritic cells.

We report the development of an optimized 50-color spectral flow cytometry panel designed for the in-depth analysis of the immune system in human blood and tissues, with the goal of maximizing the amount of information that can be collected using currently available flow cytometry platforms. We established and tested this panel using peripheral blood mononuclear cells (PBMCs), but included CD45 to enable its use for the analysis of human tissue samples. The panel contains lineage markers for all major immune cell subsets, and an extensive set of phenotyping markers focused on the activation and differentiation status of the T cell and dendritic cell (DC) compartment.

TGF-β broadly modifies rather than specifically suppresses reactivated memory CD8 T cells in a dose-dependent manner.

Transforming growth factor β (TGF-β) directly acts on naive, effector, and memory T cells to control cell fate decisions, which was shown using genetic abrogation of TGF-β signaling. TGF-β availability is altered by infections and cancer; however, the dose-dependent effects of TGF-β on memory CD8 T cell (T) reactivation are still poorly defined. We examined how activation and TGF-β signals interact to shape the functional outcome of T reactivation.

Colonic mucosal associated invariant T cells in Crohn's disease have a diverse and non-public T cell receptor beta chain repertoire.

Objectives: Mucosal-Associated Invariant T (MAIT) cells are T cells with a semi-invariant T cell receptor (TCR), recognizing riboflavin precursors presented by a non-polymorphic MR1 molecule. As these precursors are produced by the gut microbiome, we characterized the frequency, phenotype and clonality of MAIT cells in human colons with and without Crohn's disease (CD).

Methods: The transcriptome of MAIT cells sorted from blood and intestinal lamina propria cells from colectomy recipients were compared with other CD8+ T cells.

T Cell Repertoire Homogeneity and Blood-Gut Overlap in Patients With Inflammatory Bowel Disease.

Background & Aims: Inflammatory bowel disease (IBD) causes a marked increase in the number of T cells in the intestinal mucosa. Debate exists about whether these excess cells arise from local clonal proliferation or recruitment from the periphery.

Methods: CD8+ T cells were sorted from colon biopsy specimens and blood for T-cell receptor (TCR) β-chain sequencing.

TGF-β broadly modifies rather than specifically suppresses reactivated memory CD8 T cells in a dose-dependent manner.

Transforming growth factor β (TGF-β) directly acts on naïve, effector and memory T cells to control cell fate decisions, which was shown using genetic abrogation of TGF-β signaling. TGF-β availability is altered by infections and cancer, however the dose-dependent effects of TGF-β on memory CD8 T cell (T) reactivation are still poorly defined. We examined how activation and TGF-β signals interact to shape the functional outcome of T reactivation.

Extricating human tumour immune alterations from tissue inflammation.

Immunotherapies have achieved remarkable successes in the treatment of cancer, but major challenges remain. An inherent weakness of current treatment approaches is that therapeutically targeted pathways are not restricted to tumours, but are also found in other tissue microenvironments, complicating treatment. Despite great efforts to define inflammatory processes in the tumour microenvironment, the understanding of tumour-unique immune alterations is limited by a knowledge gap regarding the immune cell populations in inflamed human tissues.

MAdCAM-1 Costimulates T Cells through Integrin αβ to Cause Gene Expression Events Resembling Costimulation through CD28.

Successful treatment of inflammatory bowel disease (IBD) with the anti-integrin αβ mAb vedolizumab suggests that interaction of this integrin with addressin mucosal addressin cell adhesion molecule-1 (MAdCAM-1) is central to IBD pathogenesis. Although this was presumed to be due to an inhibition of lymphocyte trafficking to the gut, as has been observed in animal models, we report no depletion of CD4 T cells from the colonic mucosa as a consequence of vedolizumab treatment in humans, regardless of efficacy. Likewise, no upregulation of alternative trafficking mechanisms was observed as a consequence of therapy to suggest that this homeostasis is maintained in patients by a mechanistic escape from inhibition.

A regulatory T cell signature distinguishes the immune landscape of COVID-19 patients from those with other respiratory infections.

Despite recent studies of immunity to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), little is known about how the immune response against SARS-CoV-2 differs from other respiratory infections. We compare the immune signature from hospitalized SARS-CoV-2–infected patients to patients hospitalized prepandemic with influenza or respiratory syncytial virus (RSV). Our in-depth profiling indicates that the immune landscape in SARS-CoV-2 patients is largely similar to flu or RSV patients.

A differential regulatory T cell signature distinguishes the immune landscape of COVID-19 hospitalized patients from those hospitalized with other respiratory viral infections.

Unlabelled: SARS-CoV-2 infection has caused a lasting global pandemic costing millions of lives and untold additional costs. Understanding the immune response to SARS-CoV-2 has been one of the main challenges in the past year in order to decipher mechanisms of host responses and interpret disease pathogenesis. Comparatively little is known in regard to how the immune response against SARS-CoV-2 differs from other respiratory infections.

Escherichiacoli-Specific CD4+ T Cells Have Public T-Cell Receptors and Low Interleukin 10 Production in Crohn's Disease.

Background & Aims: Crohn's disease (CD) likely represents decreased immune tolerance to intestinal bacterial antigens. Most CD patients have high titers of antibodies to intestinal commensal proteins, including the outer membrane porin C (OmpC) of Escherichia coli.

Methods: By using major histocompatibility complex II tetramers, we identified an HLA-DRB1∗15:01-restricted peptide epitope of OmpC recognized by CD4+ T cells in peripheral blood mononuclear cells from HLA-DRB1∗15:01+ healthy control (HC) and CD patients.