Neutrophils are implicated in multiple homeostatic and pathological processes, but whether functional diversity requires discrete neutrophil subsets is not known. Here, we apply single-cell RNA sequencing to neutrophils from normal and inflamed mouse tissues. Whereas conventional clustering yields multiple alternative organizational structures, diffusion mapping plus RNA velocity discloses a single developmental spectrum, ordered chronologically. Termed here neutrotime, this spectrum extends from immature pre-neutrophils, largely in bone marrow, to mature neutrophils predominantly in blood and spleen. The sharpest increments in neutrotime occur during the transitions from pre-neutrophils to immature neutrophils and from mature marrow neutrophils to those in blood. Human neutrophils exhibit a similar transcriptomic pattern. Neutrophils migrating into inflamed mouse lung, peritoneum and joint maintain the core mature neutrotime signature together with new transcriptional activity that varies with site and stimulus. Together, these data identify a single developmental spectrum as the dominant organizational theme of neutrophil heterogeneity.

Increasing evidence suggests that neutrophils may participate in the regulation of adaptive immune responses, and can reach draining lymph nodes and crossprime naive T cells. The aim of this study was to identify the mechanism(s) involved in the migration of neutrophils to the draining lymph nodes. We demonstrate that a subpopulation of human and mouse neutrophils express CCR7. CCR7 is rapidly expressed at the membrane upon stimulation. In vitro, stimulated human neutrophils migrate in response to the CCR7 ligands CCL19 and CCL21. In vivo, injection of complete Freund adjuvant induces a rapid recruitment of neutrophils to the lymph nodes in wild-type mice but not in Ccr7 ؊/؊ mice. Moreover, intradermally injected interleukin-17-and granulocyte-macrophage colonystimulating factor-stimulated neutrophils from wild-type mice, but not from Ccr7 ؊/؊ mice, migrate to the draining lymph nodes. These results identify CCR7 as a chemokine receptor involved in the migration of neutrophils to the lymph nodes. IntroductionPolymorphonuclear neutrophils are the most abundant immune cells in human blood (60% of leukocytes). Every day, 10 11 neutrophils transit through the adult human circulation. 1 They are the first type of leukocytes recruited at the site of infection, where they represent the major infiltrating cells. Their migration and subsequent activation are controlled by chemokines and cytokines such as chemokine (C-X-C motif) ligand 8 (CXCL8) and tumor necrosis factor-␣ (TNF-␣). At the inflammatory site, microorganisms are phagocytosed by neutrophils, destroyed via oxygendependent or oxygen-independent mechanisms, or sequestered in extracellular traps. 1,2 Neutrophils also contain an important arsenal of microbicidal mediators. 3 Neutrophils are short-lived, terminally differentiated cells that have long been considered to be the prototypic innate immune cells and to have a restricted number of properties. 4,5 Nevertheless, recent studies have shown that the biologic properties of neutrophils are more complicated than was previously thought. Neutrophils exhibit characteristic features classically dedicated to professional antigen-presenting cells. 6,7 Under certain circumstances, neutrophils display a dendritic cell (DC)-like phenotype, as evidenced by the expression of the DC markers CD83, CD80, CD86, and major histocompatibility complex II (MHC-II) molecules. Neutrophils may present antigens in an MHC-II-dependent manner and induce the proliferation of antigen-specific T cells. [6][7][8] More recently, we reported that neutrophils cross-prime naive CD8 ϩ T lymphocytes. 9 Finally, neutrophils may also influence the polarization of antigen-specific T-cell responses. [10][11][12] Interestingly, previous studies have reported that, after the capture of antigens in the periphery, neutrophils may migrate to the lymph nodes. [13][14][15] In a murine model of Toxoplasma gondii infection, parasites are transported by neutrophils from the infection site to draining lymph nodes. 15 Similarly, after an injection of ovalbumin (O...