Selective differentiation of naïve T lymphocytes into multipotent T cells is of great interest clinically for the generation of cell-based cancer immunotherapies. Cellular differentiation depends crucially on division state and time. Here we adapt a dye-dilution assay for tracking cell proliferative history to mass cytometry, and uncouple division, time and regulatory protein expression in single naïve human T cells during their activation and expansion in a complex ex vivo milieu. Using 23 markers, we defined groups of proteins that are controlled predominantly by division state or time, and found that undivided cells account for the majority of phenotypic diversity. We next built a map of cell state changes during naïve T-cell expansion. By examining cell signaling on this map, we rationally selected ibrutinib, a BTK/ITK inhibitor administered before activation, to direct differentiation toward a T SCM -like phenotype. This method for tracing cell fate across division states and time can be broadly applied for directing cellular differentiation.

Candida albicans is an opportunistic pathogen, typically found as a benign commensal yeast living on skin and mucosa, but poised to invade injured tissue to cause local infections. In debilitated and immunocompromised individuals, C. albicans may spread to cause life-threatening systemic infections. Upon contact with serum and at body temperature, C. albicans performs a regulated switch to filamentous morphology, characterized by emergence of a germ tube from the yeast cell followed by mold-like growth of branching hyphae. The ability to switch between growth morphologies is an important virulence factor of C. albicans. To identify compounds able to inhibit hyphal morphogenesis, we screened libraries of existing drugs for inhibition of the hyphal switch under stringent conditions. Several compounds that specifically inhibited hyphal morphogenesis were identified. Chemogenomic analysis suggested an interaction with the endocytic pathway, which was confirmed by direct measurement of fluid-phase endocytosis in the presence of these compounds. These results suggest that the activity of the endocytic pathway, which is known to be particularly important for hyphal growth, represents an effective target for hyphae-inhibiting drugs.

Summary Basophils, the rarest granulocyte, play critical roles in parasite- and allergen-induced inflammation. We applied mass cytometry (CyTOF) to simultaneously asses 44 proteins to phenotype and functionally characterize neutrophils, eosinophils, and basophils from 19 healthy donors. There was minimal heterogeneity seen in eosinophils and neutrophils, but data-driven analyses revealed four unique subpopulations within phenotypically basophilic granulocytes (PBG; CD45 + HLA-DR − CD123 + ). Through CyTOF and fluorescence-activated cell sorting (FACS), we classified these four PBG subpopulations as (I) CD16 low FcεRI high CD244 high (88.5 ± 1.2%), (II) CD16 high FcεRI high CD244 high (9.1 ± 0.4%), (III) CD16 low FcεRI low CD244 low (2.3 ± 1.3), and (IV) CD16 high FcεRI low CD244 low (0.4 ± 0.1%). Prospective isolation confirmed basophilic-morphology of PBG I–III, but neutrophilic-morphology of PBG IV. Functional interrogation via IgE-crosslinking or IL-3 stimulation demonstrated that PBG I–II had significant increases in CD203c expression, whereas PBG III–IV remained unchanged compared with media-alone conditions. Thus, PBG III–IV could serve roles in non-IgE-mediated immunity. Our findings offer new perspectives in human basophil heterogeneity and the varying functional potential of these new subsets in health and disease.

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