Cellular immunity has an inherent high level of functional heterogeneity. Capturing the full spectrum of these functions requires analysis of large numbers of effector molecules from single cells. We report a microfluidic platform designed for highly multiplexed (more than ten proteins), reliable, sample-efficient (~1 × 104 cells) and quantitative measurements of secreted proteins from single cells. We validated the platform by assessment of multiple inflammatory cytokines from lipopolysaccharide (LPS)-stimulated human macrophages and comparison to standard immunotechnologies. We applied the platform toward the ex vivo quantification of T cell polyfunctional diversity via the simultaneous measurement of a dozen effector molecules secreted from tumor antigen–specific cytotoxic T lymphocytes (CTLs) that were actively responding to tumor and compared against a cohort of healthy donor controls. We observed profound, yet focused, functional heterogeneity in active tumor antigen–specific CTLs, with the major functional phenotypes quantitatively identified. The platform represents a new and informative tool for immune monitoring and clinical assessment.

SUMMARY During an infection, the body increases the output of mature immune cells to fight off the pathogen. Despite convincing evidence that hematopoietic stem and progenitor cells (HSPCs) can sense pathogens directly, how this contributes to hematopoietic cell output remains unknown. Here we have combined mouse models with a single cell proteomics platform to show that in response to toll-like receptor stimulation, short-term HSCs and multipotent progenitor cells produce copious amount of diverse cytokines through the NF-κB signaling. Interestingly, the cytokine production ability of HSPCs trumps mature immune cells in both magnitude and breadth. Among cytokines produced by HSPCs, IL-6 is a particularly important regulator of myeloid differentiation and HSPC proliferation in a paracrine manner and in mediating rapid myeloid cell recovery during neutropenia. This study has uncovered a novel property of HSPCs that enables them to convert danger signals into versatile cytokine signals for regulation of stress hematopoiesis.

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Far-red and near-infrared (NIR) fluorescent materials possessing the characteristics of strong two-photon absorption and aggregation-induced emission (AIE) as well as specific targeting capability are much-sought-after for bioimaging and therapeutic applications due to their deep penetration depth and high resolution. Herein, a series of dipolar far-red and NIR AIE luminogens with a strong push-pull effect are designed and synthesized. The obtained fluorophores display bright far-red and NIR solid-state fluorescence with a high quantum yield of up to 30%, large Stokes shifts of up to 244 nm, and large two-photon absorption cross-sections of up to 887 GM. A total of three neutral AIEgens show specific lipid droplet (LD)-targeting capability, while the one with cationic and lipophilic characteristics tends to target the mitochondria specifically. All of the molecules demonstrate good biocompatibility, high brightness, and superior photostability. They also serve as efficient two-photon fluorescence-imaging agents for the clear visualization of LDs or mitochondria in living cells and tissues with deep tissue penetration (up to 150 μm) and high contrast. These AIEgens can efficiently generate singlet oxygen upon light irradiation for the photodynamic ablation of cancer cells. All of these intriguing results prove that these far-red and NIR AIEgens are excellent candidates for the two-photon fluorescence imaging of LDs or mitochondria and organelle-targeting photodynamic cancer therapy.