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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.

Background & Aims: During chronic viral infections, the apoptosis of activated T cell elicits a CD8+ T cell response directed to those cryptic epitopes that emerge from caspase‐cleaved structural proteins. Such response directed to apoptosis‐associated epitopes (AEs) contributes to the amplification of immunopathology. Methods: Here, we have analysed through flow cytometry AE‐specific CD8+ T cells in patients with chronic hepatitis B virus (HBV) infection, naïve‐to‐treatment or undergoing nucleos(t)ide‐analogue (NUC) therapy. Results: We found that AE‐specific CD8+ T cell frequencies were significantly increased only in those NUC‐treated patients who also presented advanced hepatic fibrosis. Regulatory T cells were also expanded in those patients, and AE‐specific, but not HBV‐specific, CD8+ T cell frequency positively correlated with Treg percentages. Through multiparameter flow cytometry, multidimensionality reduction and unsupervised clustering analysis, we could identify novel subpopulations among effector memory (em) and emCD45RA+ T cell (Tem and Temra) subsets. CD8+ T cells with distinct specificities differentially populated the subpopulation map: while HBV‐specific were mostly contained in the Tem subset, AE‐specific CD8+ T cells encompassed naïve, as well as T central memory, Tem and Temra cells. Conclusion: All together, these findings indicate a link between AE‐specific CD8+ T cells and advanced liver fibrosis in patients with chronic HBV infection, and suggest that virus‐specific and AE‐specific CD8+ T cells exhibit distinct differentiation states and contribute in distinct ways to immunopathology. [ABSTRACT FROM AUTHOR]

In our study, we investigated the role of CD39 on tumor‐infiltrating CD8+ T lymphocytes (CD8+ TILs) in colorectal, head and neck and pancreatic cancers. Partially confirming recent observations correlating the CD39 expression with T‐cell exhaustion, we demonstrated a divergent functional activity in CD39+CD8+ TILs. On the one hand, CD39+CD8+ TILs (as compared to their CD39− counterparts) produced significantly lower IFN‐γ and IL‐2 amounts, expressed higher PD‐1, and inversely correlated with perforin and granzyme B expression. On the other, they displayed a significantly higher proliferative capacity ex vivo that was inversely correlated with the PD‐1 expression. Therefore, CD39+CD8+ TILs, including those co‐expressing the CD103 (a marker of T resident memory [TRM] cells), were defined as partially dysfunctional T cells that correlate with tumor patients with initial progression stages. Interestingly, our results identified for the first time a single nucleotide polymorphism (SNP rs10748643 A>G), as a genetic factor associated with CD39 expression in CD8+ TILs. Finally, we demonstrated that compounds inhibiting CD39‐related ATPases improved CD39+CD8+ T‐cell effector function ex vivo, and that CD39+CD8+ TILs displayed effective suppression function in vitro. Overall these data suggest that the SNP analysis may represent a suitable predictor of CD39+CD8+ T‐cell expression in cancer patients, and propose the modulation of CD39 as a new strategy to restore partially exhausted CD8+ TILs. What's new? CD39 is an enzyme expressed by regulatory T cells (Treg), which can suppress anti‐tumor immune responses. Recent studies have found that CD39 is also expressed by subsets of CD8+ tumor‐infiltrating lymphocytes (TILs). In the current study, the authors found that inhibiting CD39 can restore CD8+ TIL function. They also identified a SNP that may help predict dysfunctional CD39+ expression in TILs in cancer patients. [ABSTRACT FROM AUTHOR]

Thyroid hormones regulate a wide range of cellular responses, via non-genomic and genomic actions, depending on cell-specific thyroid hormone transporters, co-repressors, or co-activators. Skeletal muscle has been identified as a direct target of thyroid hormone T3, where it regulates stem cell proliferation and differentiation, as well as myofiber metabolism. However, the effects of T3 in muscle-wasting conditions have not been yet addressed. Being T3 primarily responsible for the regulation of metabolism, we challenged mice with fasting and found that T3 counteracted starvation-induced muscle atrophy. Interestingly, T3 did not prevent the activation of the main catabolic pathways, i.e., the ubiquitin-proteasome or the autophagy-lysosomal systems, nor did it stimulate de novo muscle synthesis in starved muscles. Transcriptome analyses revealed that T3 mainly affected the metabolic processes in starved muscle. Further analyses of myofiber metabolism revealed that T3 prevented the starvation-mediated metabolic shift, thus preserving skeletal muscle mass. Our study elucidated new T3 functions in regulating skeletal muscle homeostasis and metabolism in pathological conditions, opening to new potential therapeutic approaches for the treatment of skeletal muscle atrophy. [ABSTRACT FROM AUTHOR]

Besides suppressing immune responses, regulatory T cells (Tregs) maintain tissue homeostasis and control systemic metabolism. Whether iron is involved in Treg-mediated tolerance is completely unknown. Here, we showed that the transferrin receptor CD71 was upregulated on activated Tregs infiltrating human liver cancer. Mice with a Treg-restricted CD71 deficiency spontaneously developed a scurfy-like disease, caused by impaired perinatal Treg expansion. CD71-null Tregs displayed decreased proliferation and tissue-Treg signature loss. In perinatal life, CD71 deficiency in Tregs triggered hepatic iron overload response, characterized by increased hepcidin transcription and iron accumulation in macrophages. Lower bacterial diversity, and reduction of beneficial species, were detected in the fecal microbiota of CD71 conditional knockout neonates. Our findings indicate that CD71-mediated iron absorption is required for Treg perinatal expansion and is related to systemic iron homeostasis and bacterial gut colonization. Therefore, we hypothesize that Tregs establish nutritional tolerance through competition for iron during bacterial colonization after birth.

The germinal center (GC) dark zone (DZ) and light zone (LZ) regions spatially separate expansion and diversification from selection of antigen-specific B-cells to ensure antibody affinity maturation and B cell memory. The DZ and LZ differ significantly in their immune composition despite the lack of a physical barrier, yet the determinants of this polarization are poorly understood. This study provides novel insights into signals controlling asymmetric T-cell distribution between DZ and LZ regions. We identify spatially-resolved DNA damage response and chromatin compaction molecular features that underlie DZ T-cell exclusion. The DZ spatial transcriptional signature linked to T-cell immune evasion clustered aggressive Diffuse Large B-cell Lymphomas (DLBCL) for differential T cell infiltration. We reveal the dependence of the DZ transcriptional core signature on the ATR kinase and dissect its role in restraining inflammatory responses contributing to establishing an immune-repulsive imprint in DLBCL. These insights may guide ATR-focused treatment strategies bolstering immunotherapy in tumors marked by DZ transcriptional and chromatin-associated features., Competing Interests: CONFLICT OF INTERESTS ADJ has received consultancy fees from DKSH/Beigene, Roche, Gilead, Turbine Ltd, AstraZeneca, Antengene, Janssen, MSD and IQVIA; and research funding from Janssen and AstraZeneca.

In our study, we investigated the role of CD39 on tumor-infiltrating CD8 + T lymphocytes (CD8 + TILs) in colorectal, head and neck and pancreatic cancers. Partially confirming recent observations correlating the CD39 expression with T-cell exhaustion, we demonstrated a divergent functional activity in CD39 + CD8 + TILs. On the one hand, CD39 + CD8 + TILs (as compared to their CD39 - counterparts) produced significantly lower IFN-γ and IL-2 amounts, expressed higher PD-1, and inversely correlated with perforin and granzyme B expression. On the other, they displayed a significantly higher proliferative capacity ex vivo that was inversely correlated with the PD-1 expression. Therefore, CD39 + CD8 + TILs, including those co-expressing the CD103 (a marker of T resident memory [TRM] cells), were defined as partially dysfunctional T cells that correlate with tumor patients with initial progression stages. Interestingly, our results identified for the first time a single nucleotide polymorphism (SNP rs10748643 A>G), as a genetic factor associated with CD39 expression in CD8 + TILs. Finally, we demonstrated that compounds inhibiting CD39-related ATPases improved CD39 + CD8 + T-cell effector function ex vivo, and that CD39 + CD8 + TILs displayed effective suppression function in vitro. Overall these data suggest that the SNP analysis may represent a suitable predictor of CD39 + CD8 + T-cell expression in cancer patients, and propose the modulation of CD39 as a new strategy to restore partially exhausted CD8 + TILs., (© 2020 UICC.)