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.

International audienceThe classical model of hematopoiesis established in the mouse postulates that lymphoid cells originate from a founder population of common lymphoid progenitors. Here, using a modeling approach in humanized mice, we showed that human lymphoid development stemmed from distinct populations of CD127(-) and CD127(+) early lymphoid progenitors (ELPs). Combining molecular analyses with in vitro and in vivo functional assays, we demonstrated that CD127(-) and CD127(+) ELPs emerged independently from lympho-mono-dendritic progenitors, responded differently to Notch1 signals, underwent divergent modes of lineage restriction, and displayed both common and specific differentiation potentials. Whereas CD127(-) ELPs comprised precursors of T cells, marginal zone B cells, and natural killer (NK) and innate lymphoid cells (ILCs), CD127(+) ELPs supported production of all NK cell, ILC, and B cell populations but lacked T potential. On the basis of these results, we propose a "two-family" model of human lymphoid development that differs from the prevailing model of hematopoiesis

BackgroundThe cancer-testis antigen MAGE-A4 is an attractive target for T-cell-based immunotherapy, especially for indications with unmet clinical need like non-small cell lung or triple-negative breast cancer.MethodsAn unbiased CD137-based sorting approach was first used to identify an immunogenic MAGE-A4-derived epitope (GVYDGREHTV) that was properly processed and presented on human leukocyte antigen (HLA)-A2 molecules encoded by the HLA-A*02:01 allele. To isolate high-avidity T cells via subsequent multimer sorting, an in vitro priming approach using HLA-A2-negative donors was conducted to bypass central tolerance to this self-antigen. Pre-clinical parameters of safety and activity were assessed in a comprehensive set of in vitro and in vivo studies.ResultsA MAGE-A4-reactive, HLA-A2-restricted T-cell receptor (TCR) was isolated from primed T cells of an HLA-A2-negative donor. The respective TCR-T-cell (TCR-T) product bbT485 was demonstrated pre-clinically to have a favorable safety profile and superior in vivo potency compared with TCR-Ts expressing a TCR derived from a tolerized T-cell repertoire to self-antigens. This natural high-avidity TCR was found to be CD8 co-receptor independent, allowing effector functions to be elicited in transgenic CD4+ T helper cells. These CD4+ TCR-Ts supported an anti-tumor response by direct killing of MAGE-A4-positive tumor cells and upregulated hallmarks associated with helper function, such as CD154 expression and release of key cytokines on tumor-specific stimulation.ConclusionThe extensive pre-clinical assessment of safety and in vivo potency of bbT485 provide the basis for its use in TCR-T immunotherapy studies. The ability of this non-mutated high-avidity, co-receptor-independent TCR to activate CD8+ and CD4+ T cells could potentially provide enhanced cellular responses in the clinical setting through the induction of functionally diverse T-cell subsets that goes beyond what is currently tested in the clinic.

21 ] i may in turn activate TRPA 1 leading to a greater rise in [Ca 21 ] i . We also recognize that these studies were performed in vitro, which may not fully recapitulate what occurs in vivo. Finally, studies using primary bronchial epithelial cells from deceased donors are intrinsically limited by the availability of donor cells, especially in the pediatric age group. Additionally, history of atopic status and lung function is frequently not available, as well as comorbidities and cause of death. Thus, it is possible that the age effect could be explained by other factors and future studies will need to be done using cells from identified samples from patients with documented asthma status and severity.In conclusion, the results of this study show for the first time that lower airway epithelium from asthmatic children displays elevated basal TRPV 1 activity when compared with that of nonasthmatic controls. We have also shown that RSV infection of epithelial cells from asthmatic children-but not from adults-leads to an increase in overall TRPV 1 activation. Pharmacological inhibition of TRPV 1 may lead in the future to strategies that might reduce the impact of RSV infections in both asthmatic and nonasthmatic children.

BackgroundThe cancer-testis antigen MAGE-A4 is an attractive target for T cell-based immunotherapy, especially for indications with unmet clinical need like non-small-cell lung carcinoma or triple-negative breast cancer. Overcoming high tumor burden using adoptive transfer of T cells modified to express a transgenic T cell receptor (TCR) demands optimal recognition of the corresponding target on tumor cells by the TCR-modified T cells (TCR-Ts). Here we describe the isolation and pre-clinical characterization of high avidity TCR-Ts expressing a human leucocyte antigen (HLA)-A*02:01-restricted MAGE-A4-specific TCR that is fully functional in T cells irrespective of CD4 or CD8 co-receptor expression.MethodsAn unbiased CD137-based sorting approach was first used to identify an immunogenic MAGE-A4-derived candidate epitope that was properly processed and presented on HLA-A2 molecules encoded by the HLA-A*02:01 allele. To isolate high avidity T cells via subsequent multimer sorting, an in vitro priming approach using HLA-A2-negative donors (allogeneic-HLA-restricted priming approach) was conducted to bypass central tolerance to this self-antigen. Pre-clinical parameters of safety and activity were assessed in a comprehensive set of in vitro and in vivo studies of the lead TCR candidate derived from a selected T cell clone.ResultsA TCR recognizing the MAGE-A4-derived decapeptide GVYDGREHTV was isolated from primed T cells of a non-tolerant HLA-A2-negative donor. The respective TCR-T cell product bbT485, expressing the lead TCR in T cells from healthy donors, was demonstrated pre-clinically to have a favorable safety profile and superior in vivo potency compared to TCR-Ts made using a TCR derived from an HLA-A2-positive donor bearing a tolerized T cell repertoire to self-antigens. The natural high avidity allogeneic (allo)-derived TCR was found to be CD8 co-receptor-independent, allowing effector functions to be elicited in transgenic CD4+ T helper cells. These CD4+ TCR-T cells not only supported an anti-tumor response by direct killing of MAGE-A4-positive tumor cells, but also upregulated hallmarks associated with helper function, such as CD154 expression and release of key cytokines upon tumor-specific stimulation.ConclusionsThe extensive pre-clinical assessment of safety and in vivo potency of this non-mutated high avidity, CD8 co-receptor-independent, MAGE-A4-specific HLA-A2 restricted TCR provide the basis for its use in clinical TCR-T immunotherapy studies. The ability of this co-receptor-independent TCR to activate all transduced T cells (irrespective of CD4 or CD8 expression) could potentially provide enhanced cellular responses in the clinical setting through the induction of functionally diverse T cell subsets that goes beyond what is currently tested in the clinic.