These guidelines are a consensus work of a considerable number of members of the immunology and flow cytometry community. They provide the theory and key practical aspects of flow cytometry enabling immunologists to avoid the common errors that often undermine immunological data. Notably, there are comprehensive sections of all major immune cell types with helpful Tables detailing phenotypes in murine and human cells. The latest flow cytometry techniques and applications are also described, featuring examples of the data that can be generated and, importantly, how the data can be analysed. Furthermore, there are sections detailing tips, tricks and pitfalls to avoid, all written and peer‐reviewed by leading experts in the field, making this an essential research companion.

An open-label, first-in-human phase 1/2 study is being conducted to evaluate the safety and efficacy of pancreatic endoderm cells (PECs) implanted in non-immunoprotective macroencapsulation devices for the treatment of type 1 diabetes. We report an analysis on 1 year of data from the first cohort of 15 patients from a single trial site that received subcutaneous implantation of cell products combined with an immunosuppressive regimen. Implants were well tolerated with no teratoma formation or severe graft-related adverse events. After implantation, patients had increased fasting C-peptide levels and increased glucose-responsive C-peptide levels and developed mixed meal-stimulated C-peptide secretion. There were immunosuppression-related transient increases in circulating regulatory T cells, PD1 high T cells, and IL17A + CD4 + T cells. Explanted grafts contained cells with a mature b cell phenotype that were immunoreactive for insulin, islet amyloid polypeptide, and MAFA. These data, and associated findings (Shapiro et al., 2021), are the first reported evidence of meal-regulated insulin secretion by differentiated stem cells in patients.*No ACR sample was collected, but patients had negative protein on a urine dipstick test. BMI, Body mass index; ACR, albumin-creatinine ratio; MDI, multiple daily injections. ll Clinical and Translational Report

The third edition of Flow Cytometry Guidelines provides the key aspects to consider when performing flow cytometry experiments and includes comprehensive sections describing phenotypes and functional assays of all major human and murine immune cell subsets. Notably, the Guidelines contain helpful tables highlighting phenotypes and key differences between human and murine cells. Another useful feature of this edition is the flow cytometry analysis of clinical samples with examples of flow cytometry applications in the context of autoimmune diseases, cancers as well as acute and chronic infectious diseases. Furthermore, there are sections detailing tips, tricks and pitfalls to avoid. All sections are written and peer-reviewed by leading flow cytometry experts and immunologists, making this edition an essential and state-of-the-art handbook for basic and clinical researchers.

Pulmonary gammadelta T cells protect the lung and its functions, but little is known about their distribution in this organ and their relationship to other pulmonary cells. We now show that gammadelta and alphabeta T cells are distributed differently in the normal mouse lung. The gammadelta T cells have a bias for nonalveolar locations, with the exception of the airway mucosa. Subsets of gammadelta T cells exhibit further variation in their tissue localization. gammadelta and alphabeta T cells frequently contact other leukocytes, but they favor different cell-types. The gammadelta T cells show an intrinsic preference for F4/80+ and major histocompatibility complex class II+ leukocytes. Leukocytes expressing these markers include macrophages and dendritic cells, known to function as sentinels of airways and lung tissues. The continuous interaction of gammadelta T cells with these sentinels likely is related to their protective role.

Regulatory T cells (Tregs) are suppressive T cells that have an essential role in maintaining the balance between immune activation and tolerance. Their development, either in the thymus, periphery, or experimentally in vitro, and stability and function all depend on the right mix of environmental stimuli. This review focuses on the effects of cytokines, metabolites, and the microbiome on both human and mouse Treg biology. The role of cytokines secreted by innate and adaptive immune cells in directing Treg development and shaping their function is well established. New and emerging data suggest that metabolites, such as retinoic acid, and microbial products, such as short-chain fatty acids, also have a critical role in guiding the functional specialization of Tregs. Overall, the complex interaction between distinct environmental stimuli results in unique, and in some cases tissue-specific, tolerogenic environments. Understanding the conditions that favor Treg induction, accumulation, and function is critical to defining the pathophysiology of many immune-mediated diseases and to developing new therapeutic interventions.

Human Ag-specific CD4 + T cells can be detected by their dual expression of CD134 (OX40) and CD25 after a 44 hours stimulation with cognate Ag. We show that surface expression of CD39 on Ag-specific cells consistently identifies a substantial population of CD4 + CD25 + CD134 + CD39 + T cells that have a Treg-cell-like phenotype and mostly originate from bulk memory CD4 + CD45RO + CD127 low CD25 high CD39 + Treg cells. Viable, Ag-specific CD25 + CD134 + CD39 + T cells could be expanded in vitro as cell lines and clones, and retained high Forkhead Box Protein 3, CTLA-4 and CD39 expression, suppressive activity and Ag specificity. We also utilised this combination of cell surface markers to measure HIV-Gag responses in HIV + patients before and after anti-retroviral therapy (ART). Interestingly, we found that the percentage of CD39 − cells within baseline CD4 + T-cell responses to HIV-Gag was negatively correlated with HIV viral load pre-ART and positively correlated with CD4 + T-cell recovery over 96 weeks of ART. Collectively, our data show that Ag-specific CD4 + CD25 + CD134 + CD39 + T cells are highly enriched for Treg cells, form a large component of recall responses and maintain a Treg-cell-like phenotype upon in vitro expansion. Identification and isolation of these cells enables the role of Treg cells in memory responses to be further defined and provides a development pathway for novel therapeutics.Keywords: Antigen-specific T cells r CD25 r CD39 r CD134 r FOXP3 r OX40 r Treg cell Additional supporting information may be found in the online version of this article at the publisher's web-site Correspondence: Laura Cook e-mail: lcook@kirby.unsw.edu.au * These authors contributed equally to this work. The current gold standard marker for the study of Treg cells is Forkhead Box Protein 3 (FOXP3), a constitutively expressed nuclear transcription factor, critical for Treg-cell survival and suppressive function [3]. The key limitation of this marker is that viable cells cannot be isolated, as staining for this intracellular protein involves cell permeabilisation. Also, in the context of activation, this marker loses specificity due to transient, activationinduced up-regulation of FOXP3 in human non-Treg cells [4,5]. An alternative, widely used definition of Treg cells is the combined cell surface expression of high levels of CD25 (IL-2Rα) and low levels of CD127 (IL-7Rα) [6]. Whilst >85% of CD127 low CD25 high Treg cells express FOXP3 and are suppressive ex vivo [6], activated Treg cells cannot be isolated with these markers due to CD127 down-regulation, and CD25 up-regulation, on other subsets of CD4 + T cells following activation by cognate .In 2007, it was shown that murine Treg cells co-express the ectoenzymes CD39 and CD73 [10]. These ectonucleotidases work in concert to convert adenosine triphosphate (ATP), adenosine diphosphate and adenosine monophosphate to immunosuppressive adenosine, which appears to play a role in the suppressive repertoire of Treg cells [10]. Apart from contributing to adenosine produ...

This study provides the first estimation of FOXP3CD39 Treg cell frequency within circulating gluten-specific CD4 T cells after oral gluten challenge of patients with celiac disease. FOXP3CD39 Treg cells comprised a major proportion of all circulating gluten-specific CD4 T cells but had impaired suppressive function, indicating that Treg cell dysfunction might be a key contributor to disease pathogenesis.

Airway hyperresponsiveness (AHR), a hallmark of asthma and several other diseases, can be modulated by γδ T cells. In mice sensitized and challenged with OVA, AHR depends on allergen-specific αβ T cells; but Vγ1+ γδ T cells spontaneously enhance AHR, whereas Vγ4+ γδ T cells, after being induced by airway challenge, suppress AHR. The activity of these γδ T cell modulators is allergen nonspecific, and how they develop is unclear. We now show that CD8 is essential for the development of both the AHR suppressor and enhancer γδ T cells, although neither type needs to express CD8 itself. Both cell types encounter CD8-expressing non-T cells in the spleen, and their functional development in an otherwise CD8-negative environment can be restored with transferred spleen cell preparations containing CD8+ dendritic cells (DCs), but not CD8+ T cells or CD8− DCs. Our findings suggest that CD8+ DCs in the lymphoid tissues enable an early step in the development of γδ T cells through direct cell contact. DC-expressed CD8 might take part in this interaction.