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As the largest organ of the body, the skin is a key barrier tissue with specialized structures where ongoing immune surveillance is critical for protecting the body from external insults. The innate immune system acts as first-responders in a coordinated manner to react to injury or infections, and recent developments in intravital imaging techniques have made it possible to delineate dynamic immune cell responses in a spatiotemporal manner. We review here key studies involved in understanding neutrophil, dendritic cell and macrophage behavior in skin and further discuss how this knowledge collectively highlights the importance of interactions and cellular functions in a systems biology manner. Furthermore, we will review emerging imaging technologies such as high-content proteomic screening, spatial transcriptomics and three-dimensional volumetric imaging and how these techniques can be integrated to provide a systems overview of the immune system that will further our current knowledge and lead to potential exciting discoveries in the upcoming decades.

Circulating Ly6Chi monocytes often undergo cellular death upon exhaustion of their antibacterial effector functions, which limits their capacity for subsequent macrophage differentiation. This shrouds the understanding on how the host replaces the tissue-resident macrophage niche effectively during bacterial invasion to avert infection morbidity. Here, we show that proliferating transitional premonocytes (TpMos), an immediate precursor of mature Ly6Chi monocytes (MatMos), were mobilized into the periphery in response to acute bacterial infection and sepsis. TpMos were less susceptible to apoptosis and served as the main source of macrophage replenishment when MatMos were vulnerable toward bacteria-induced cellular death. Furthermore, TpMo and its derived macrophages contributed to host defense by balancing the proinflammatory cytokine response of MatMos. Consequently, adoptive transfer of TpMos improved the survival outcome of lethal sepsis. Our findings hence highlight a protective role for TpMos during bacterial infections and their contribution toward monocyte-derived macrophage heterogeneity in distinct disease outcomes. Agency for Science, Technology and Research (A*STAR) National Research Foundation (NRF) Published version This research was funded by Singapore Immunology Network (SIgN) core funding and A*STAR, Singapore. F.G. is an EMBO YIP awardee and is supported by SIgN core funding and Singapore National Research Foundation Investigatorship (NRF2016NRF-NRF1001-02). L.G.N. is supported by SIgN core funding. S.Z.C. is supported by the Ministry of Health, Singapore’s National Medical Research Council under its Open Fund-Young Individual Research Grant (OFYIRG17may036), and A*STAR Career Development Award (192D8043). Y.C.T. is a graduate scholar supported by the Ministry of Education, Singapore. M.Y.C. is a graduate scholar supported by the SIgN-NTU-NUS scholarship, Singapore. SIgN Flow Cytometry facility is supported by National Research Foundation (NRF) Singapore under Shared Infrastructure Support (SIS) (NRF2017_SISFP09).

Tissue‐resident macrophages in white adipose tissue (WAT) dynamically adapt to the metabolic changes of their microenvironment that are often induced by excess energy intake. Currently, the exact contribution of these macrophages in obesity‐driven WAT remodeling remains controversial. Here, using a transgenic CD169‐DTR mouse strain, we provide new insights into the interplay between CD169+ adipose tissue macrophages (ATMs) and their surrounding WAT microenvironment. Using targeted in vivo ATM ablation followed by transcriptional and metabolic WAT profiling, we found that ATMs protect WAT from the excessive pathological remodeling that occurs during obesity. As obesity progresses, ATMs control not only vascular integrity, adipocyte function, and lipid and metabolic derangements but also extracellular matrix accumulation and resultant fibrosis in the WAT. The protective role of ATMs during obesity‐driven WAT dysfunction supports the notion that ATMs represent friends, rather than foes, as has previously assumed., This study reveals that resident macrophages in the adipose tissue are important regulatory cells that restrain tissue dysregulation caused by obesity.

Pressure ulcers are a chronic problem for patients or the elderly who require extended periods of bed rest. The formation of ulcers is due to repeated cycles of ischemia-reperfusion (IR), which initiates an inflammatory response. Advanced ulcers disrupt the skin barrier, resulting in further complications. To date, the immunological aspect of skin IR has been understudied, partly due to the complexity of the skin immune cells. Through a combination of mass cytometry, confocal imaging and intravital multiphoton imaging, this study establishes a workflow for multidimensionality single cell analysis of skin myeloid cell responses in the context of IR injury with high spatiotemporal resolution. The data generated has provided us with previously uncharacterized insights into the distinct cellular behavior of resident dendritic cells (DCs) and recruited neutrophils post IR. Of interest, we observed a drop in DDC numbers in the IR region, which was subsequently replenished 48h post IR. More importantly, in these cells, we observe an attenuated response to repeated injuries, which may have implications in the subsequent wound healing process. Agency for Science, Technology and Research (A*STAR) National Medical Research Council (NMRC) We thank Dr. Michel Nussenzweig and Dr. Thomas Graf for providing us with the CD11c‐EYFP and LysM‐ eGFP mice, respectively. This research was funded by SIgN core funding, A*STAR, Singapore and National Health and Medical Research Council, Australia (1106439 to W.W. and L.G. NG).

Macrophages play an important role in structural cardiac remodeling and the transition to heart failure following myocardial infarction (MI). Previous research has focused on the impact of blood-derived monocytes on cardiac repair. Here we examined the contribution of resident cavity macrophages located in the pericardial space adjacent to the site of injury. We found that disruption of the pericardial cavity accelerated maladaptive post-MI cardiac remodeling. Gata6+ macrophages in mouse pericardial fluid contributed to the reparative immune response. Following experimental MI, these macrophages invaded the epicardium, lost Gata6 expression, but continued to perform anti-fibrotic functions. Loss of this specialized macrophage population enhanced interstitial fibrosis after ischemic injury. Gata6+ macrophages were present in human pericardial fluid, supporting the notion that this reparative function is relevant in human disease. Our findings uncover an immune cardioprotective role for the pericardial tissue compartment and argue for the reevaluation of surgical procedures that remove the pericardium.

Granulocyte-monocyte progenitors (GMPs) have been previously defined for their potential to generate various myeloid progenies such as neutrophils and monocytes. Although studies have proposed lineage heterogeneity within GMPs, it is unclear if committed progenitors already exist among these progenitors and how they may behave differently during inflammation. By combining single-cell transcriptomic and proteomic analyses, we identified the early committed progenitor within the GMPs responsible for the strict production of neutrophils, which we designate as proNeu1. Our dissection of the GMP hierarchy led us to further identify a previously unknown intermediate proNeu2 population. Similar populations could be detected in human samples. proNeu1s, but not proNeu2s, selectively expanded during the early phase of sepsis at the expense of monocytes. Collectively, our findings help shape the neutrophil maturation trajectory roadmap and challenge the current definition of GMPs.

Monocytes are a subset of cells that are categorized together with dendritic cells (DCs) and macrophages in the mononuclear phagocyte system (MPS). Despite sharing several phenotypic and functional characteristics with MPS cells, monocytes are unique cells with the ability to function as both precursor and effector cells in their own right. Before the development of hematopoietic stem cells (HSCs) in utero, monocytes are derived from erythro-myeloid precursors (EMPs) in the fetal liver that are important for populating the majority of tissue resident macrophages. After birth, monocytes arise from bone marrow (BM)-derived HSCs and are released into the circulation upon their maturation, where they survey peripheral tissues and maintain endothelial integrity. Upon sensing of microbial breaches or inflammatory stimuli, monocytes migrate into tissues where their plasticity allows them to differentiate into cells that resemble macrophages or DCs according to the environmental niche. Alternatively, they may also migrate into tissues in the absence of inflammation and remain in an undifferentiated state where they perform homeostatic roles. As monocytes are typically on the move, the availability of intravital imaging approaches has provided further insights into their trafficking patterns in distinct tissue compartments. In this review, we outline the importance of understanding their functional behavior in the context of tissue compartments, and how these studies may contribute towards improved vaccine and future therapeutic strategies. NMRC (Natl Medical Research Council, S’pore) Published version

Neutrophils eliminate pathogens efficiently but can inflict severe damage to the host if they over-activate within blood vessels. It is unclear how immunity solves the dilemma of mounting an efficient anti-microbial defense while preserving vascular health. Here, we identify a neutrophil-intrinsic program that enabled both. The gene Bmal1 regulated expression of the chemokine CXCL2 to induce chemokine receptor CXCR2-dependent diurnal changes in the transcriptional and migratory properties of circulating neutrophils. These diurnal alterations, referred to as neutrophil aging, were antagonized by CXCR4 (C-X-C chemokine receptor type 4) and regulated the outer topology of neutrophils to favor homeostatic egress from blood vessels at night, resulting in boosted anti-microbial activity in tissues. Mice engineered for constitutive neutrophil aging became resistant to infection, but the persistence of intravascular aged neutrophils predisposed them to thrombo-inflammation and death. Thus, diurnal compartmentalization of neutrophils, driven by an internal timer, coordinates immune defense and vascular protection. Neutrophils display circadian oscillations in numbers and phenotype in the circulation. Adrover and colleagues now identify the molecular regulators of neutrophil aging and show that genetic disruption of this process has major consequences in immune cell trafficking, anti-microbial defense, and vascular health., This study was supported by Intramural grants from A∗STAR to L.G.N., BES-2013-065550 to J.M.A., BES-2010-032828 to M.C.-A, and JCI-2012-14147 to L.A.W (all from Ministerio de Economía, Industria y Competitividad; MEIC). Additional MEIC grants were SAF2014-61993-EXP to C.L.-R.; SAF2015-68632-R to M.A.M. and SAF-2013-42920R and SAF2016-79040Rto D.S. D.S. also received 635122-PROCROP H2020 from the European Commission and ERC CoG 725091 from the European Research Council (ERC). ERC AdG 692511 PROVASC from the ERC and SFB1123-A1 from the Deutsche Forschungsgemeinschaft were given to C.W.; MHA VD1.2/81Z1600212 from the German Center for Cardiovascular Research (DZHK) was given to C.W. and O.S.; SFB1123-A6 was given to O.S.; SFB914-B08 was given to O.S. and C.W.; and INST 211/604-2, ZA 428/12-1, and ZA 428/13-1 were given to A.Z. This study was also supported by PI12/00494 from Fondo de Investigaciones Sanitarias (FIS) to C.M.; PI13/01979, Cardiovascular Network grant RD 12/0042/0054, and CIBERCV to B.I.; SAF2015-65607-R, SAF2013-49662-EXP, and PCIN-2014-103 from MEIC; and co-funding by Fondo Europeo de Desarrollo Regional (FEDER) to A.H. The CNIC is supported by the MEIC and the Pro CNIC Foundation and is a Severo Ochoa Center of Excellence (MEIC award SEV-2015-0505).

Deposition of immune complexes (ICs) in tissues triggers acute inflammatory pathology characterized by massive neutrophil influx leading to edema and hemorrhage, and is especially associated with vasculitis of the skin, but the mechanisms that regulate this type III hypersensitivity process remain poorly understood. Here, using a combination of multiphoton intravital microscopy and genomic approaches, we re-examined the cutaneous reverse passive Arthus reaction and observed that IC-activated neutrophils underwent transmigration, triggered further IC formation, and transported these ICs into the interstitium, whereas neutrophil depletion drastically reduced IC formation and ameliorated vascular leakage in vivo. Thereafter, we show that these neutrophils expressed high levels of CXCL2, which further amplified neutrophil recruitment and activation in an autocrine and/or paracrine manner. Notably, CXCL1 expression was restricted to tissue-resident cell types, but IC-activated neutrophils may also indirectly, via soluble factors, modulate macrophage CXCL1 expression. Consistent with their distinct cellular origins and localization, only neutralization of CXCL2 but not CXCL1 in the interstitium effectively reduced neutrophil recruitment. In summary, our study establishes that neutrophils are able to self-regulate their own recruitment and responses during IC-mediated inflammation through a CXCL2-driven feed forward loop.

Monocytes, dendritic cells (DCs) and macrophages have been classically categorized into the mononuclear phagocyte system (MPS) based on their similar functional and phenotypic characteristics. While an increasing amount of research has revealed substantial ontogenic and functional differences among these cells, the reasons behind their heterogeneity and strategic positioning in specific niches throughout the body are yet to be fully elucidated. In this review, we outline how recent advances in intravital imaging studies have dissected this phenomenon and have allowed us to appreciate how MPS cells exploit their regional niches to specialize and maximize their functional properties. Understanding their cellular behavior in each of their specialized microenvironment will eventually allow us to target specific cells and their behavioral patterns for improved vaccine and therapeutic purposes.

Neutrophils are specialized innate cells that require constant replenishment from proliferative bone marrow (BM) precursors as a result of their short half-life. Although it is established that neutrophils are derived from the granulocyte-macrophage progenitor (GMP), the differentiation pathways from GMP to functional mature neutrophils are poorly defined. Using mass cytometry (CyTOF) and cell-cycle-based analysis, we identified three neutrophil subsets within the BM: a committed proliferative neutrophil precursor (preNeu) which differentiates into non-proliferating immature neutrophils and mature neutrophils. Transcriptomic profiling and functional analysis revealed that preNeu require the C/EBPε transcription factor for their generation from the GMP, and their proliferative program is substituted by a gain of migratory and effector function as they mature. preNeus expand under microbial and tumoral stress, and immature neutrophils are recruited to the periphery of tumor-bearing mice. In summary, our study identifies specialized BM granulocytic populations that ensure supply under homeostasis and stress responses. We thank all members of L.G.N laboratory, the SIgN (Singapore Immunology Network) flow-cytometry team, the SIgN functional genomics team for their assistance with transcriptomics, and the SIgN mouse core facility for their technical help and support. This research was funded by SIgN core funding, A*STAR (Agency for Science, Technology and Research), Singapore. Sí

In adult mammals, lymphatic vessels have been shown to respond to their environment by undergoing lymphangiogenesis, the formation of new lymphatic vessels from preexisting ones. Accumulating experimental and preclinical studies demonstrate that lymphangiogenesis is associated with many inflammatory diseases and may represent an attractive therapeutic target for inflammatory diseases. Thus, a better understanding of how lymphangiogenesis is regulated and contribution to inflammation is critical and may benefit clinical research targeting chronic inflammatory diseases. This review discusses the biological functions of lymphangiogenesis during inflammation and our current understanding of the key cellular players that can either support or limit lymphangiogenesis. Current data suggest that the context and time frame in which lymphangiogenesis occurs will determine its impact on the course of inflammation.

CXCR4 identifies an immobilized BM precursor (i.e., transitional premonocyte [TpMo]) that proliferates and replenishes mature Ly6Chi monocytes in mice and humans. Upon entering the circulation, CXCR4 governs monocyte margination in the lung vasculature., It is well established that Ly6Chi monocytes develop from common monocyte progenitors (cMoPs) and reside in the bone marrow (BM) until they are mobilized into the circulation. In our study, we found that BM Ly6Chi monocytes are not a homogenous population, as current data would suggest. Using computational analysis approaches to interpret multidimensional datasets, we demonstrate that BM Ly6Chi monocytes consist of two distinct subpopulations (CXCR4hi and CXCR4lo subpopulations) in both mice and humans. Transcriptome studies and in vivo assays revealed functional differences between the two subpopulations. Notably, the CXCR4hi subset proliferates and is immobilized in the BM for the replenishment of functionally mature CXCR4lo monocytes. We propose that the CXCR4hi subset represents a transitional premonocyte population, and that this sequential step of maturation from cMoPs serves to maintain a stable pool of BM monocytes. Additionally, reduced CXCR4 expression on monocytes, upon their exit into the circulation, does not reflect its diminished role in monocyte biology. Specifically, CXCR4 regulates monocyte peripheral cellular activities by governing their circadian oscillations and pulmonary margination, which contributes toward lung injury and sepsis mortality. Together, our study demonstrates the multifaceted role of CXCR4 in defining BM monocyte heterogeneity and in regulating their function in peripheral tissues.

Background We previously reported that prior nasal administration of highly attenuated Bordetella pertussis BPZE1 provides effective and sustained protection against lethal challenge with influenza A viruses. The protective effect was mediated by suppressing the production of major pro-inflammatory mediators. To further explore the anti-inflammatory properties of BPZE1, we investigated the effect of BPZE1 nasal pretreatment on two mouse models of allergic disease, allergic airway inflammation, and contact hypersensitivity (CHS). Methods Allergic reactions were induced in mice nasally pretreated with live attenuated BPZE1 bacteria using the ovalbumin (OVA)-induced allergic airway inflammation and dinitrochlorobenzene (DNCB)-induced CHS models. Results Prior BPZE1 nasal treatment suppressed OVA-induced lung inflammation and inflammatory cell recruitment and significantly reduced IgE levels and cytokine production. Similarly, BPZE1 nasal pretreatment markedly inhibited ear swelling, skin inflammation, and production of pro-inflammatory cytokines in the DNCB-induced CHS model. For both models, we showed that BPZE1 pretreatment does not affect the sensitization phase. Upon challenge, BPZE1 pretreatment selectively reduced the level of cytokines whose production is increased and did not affect the basal level of other cytokines. Together, our observations suggest that BPZE1 pretreatment specifically targets those cytokine-producing effector cells that are recruited and involved in the inflammatory reaction. Conclusion Our study demonstrates the broad anti-inflammatory properties of the attenuated B. pertussis BPZE1 vaccine candidate and supports its development as a promising agent to prevent and/or treat allergic diseases.

Monocytes are innate immune cells that play critical roles in inflammation and immune defense. A better comprehension of how monocytes are mobilized and recruited is fundamental to understand their biologic role in disease and steady state. The BM represents a major “checkpoint” for monocyte homeostasis, as it is the primary site for their production and release. Our study determined that the Cx3cr1gfp/+ mouse strain is currently the most ideal model for the visualization of monocyte behavior in the BM by multiphoton intravital microscopy. However, we observed that DCs are also labeled with high levels of GFP and thus, interfere with the accuracy of monocyte tracking in vivo. Hence, we generated a Cx3cr1gfp/+Flt3L−/− reporter mouse and showed that whereas monocyte numbers were not affected, DC numbers were reduced significantly, as DCs but not monocytes depend on Flt3 signaling for their development. We thus verified that mobilization of monocytes from the BM in Cx3cr1gfp/+Flt3L−/− mice is intact in response to LPS. Collectively, our study demonstrates that the Cx3cr1gfp/+Flt3L−/− reporter mouse model represents a powerful tool to visualize monocyte activities in BM and illustrates the potential of a Cx3cr1gfp/+-based, multifunctionality fluorescence reporter approach to dissect monocyte function in vivo.

Lymphangiogenesis is an important physiological response to inflammatory insult, acting to limit inflammation. Macrophages, dendritic cells, and lymphocytes are known to drive lymphangiogenesis. In this study, we show that neutrophils recruited to sites of inflammation can also coordinate lymphangiogenesis. In the absence of B cells, intranodal lymphangiogenesis induced during prolonged inflammation as a consequence of immunization is dependent on the accumulation of neutrophils. When neutrophils are depleted in wild-type mice developing skin inflammation in response to immunization or contact hypersensitization, lymphangiogenesis is decreased and local inflammation is increased. We demonstrate that neutrophils contribute to lymphangiogenesis primarily by modulating vascular endothelial growth factor (VEGF)-A bioavailability and bioactivity and, to a lesser extent, secreting VEGF-D. We further show that neutrophils increased VEGF-A bioavailability and bioactivity via the secretion of matrix metalloproteinases 9 and heparanase. Together, these findings uncover a novel function for neutrophils as organizers of lymphangiogenesis during inflammation.

Despite increasing studies targeted at host-pathogen interactions, vector-borne diseases remain one of the largest economic health burdens worldwide. Such diseases are vectored by hematophagous arthropods that deposit pathogens into the vertebrate host’s skin during a blood meal. These pathogens spend a substantial amount of time in the skin that allows for interaction with cutaneous immune cells, suggesting a window of opportunity for development of vaccine strategies. In particular, the recent availability of intravital imaging approaches has provided further insights into immune cell behavior in living tissues. Here, we discuss how such intravital imaging studies have contributed to our knowledge of cutaneous immune cell behavior and specifically, toward pathogen and tissue trauma from the arthropod bite. We also suggest future imaging approaches that may aid in better understanding of the complex interplay between arthropod-vectored pathogens and cutaneous immunity that could lead to improved therapeutic strategies. Published version

Monocytes and their derived cells have critical roles in inflammation and immune defense. However, their function in skin diseases such as allergic contact dermatitis remains poorly defined. Using a model of contact hypersensitivity (CHS) toward 2,4-dinitrochlorobenzene, we show that Ly6C+ CD11b+ monocytic cells participate in the pathophysiology of CHS and their accumulation is regulated by effector CD8 T cells. These Ly6C+ CD11b+ monocytic cells are the primary contributors of tumor necrosis factor-α (TNF-α) and inducible nitric oxide synthase (iNOS) and derive from Ly6C(hi)CCR2+ monocytes, as they were absent in non-inflamed skin and accumulate as a consequence of inflammation in a C-C chemokine receptor type 2 (CCR2)-dependent manner. Importantly, CCR2(-/-) mice, or wild-type mice depleted of monocytes via clodronate liposomes, display a marked decrease in TNF-α and iNOS expression accompanied by attenuated skin inflammation. Using transgenic mice and antibody depletion, we show that effector CD8 T cells regulate the accumulation of Ly6C+ CD11b+ monocytic cells through IL-17 and activate them for TNF-α and iNOS through IFN-γ. CD8 T cell-derived IFN-γ was also critical for the accumulation of the major histocompatibility complex II-expressing Ly6C+ CD11b+ subset, which expressed intermediate levels of CD11c and costimulatory molecules. Taken together, our findings provide further insight into the pathophysiology of allergic contact dermatitis by showing that CD8 T cells regulate the inflammatory cascade through TNF/iNOS-expressing Ly6C+ CD11b+ monocytic cells.

The CXCR4 antagonist plerixafor augments frequency of circulating neutrophils via release from the lung and prevents neutrophil homing to the bone marrow., Blood neutrophil homeostasis is essential for successful host defense against invading pathogens. Circulating neutrophil counts are positively regulated by CXCR2 signaling and negatively regulated by the CXCR4–CXCL12 axis. In particular, G-CSF, a known CXCR2 signaler, and plerixafor, a CXCR4 antagonist, have both been shown to correct neutropenia in human patients. G-CSF directly induces neutrophil mobilization from the bone marrow (BM) into the blood, but the mechanisms underlying plerixafor-induced neutrophilia remain poorly defined. Using a combination of intravital multiphoton microscopy, genetically modified mice and novel in vivo homing assays, we demonstrate that G-CSF and plerixafor work through distinct mechanisms. In contrast to G-CSF, CXCR4 inhibition via plerixafor does not result in neutrophil mobilization from the BM. Instead, plerixafor augments the frequency of circulating neutrophils through their release from the marginated pool present in the lung, while simultaneously preventing neutrophil return to the BM. Our study demonstrates for the first time that drastic changes in blood neutrophils can originate from alternative reservoirs other than the BM, while implicating a role for CXCR4–CXCL12 interactions in regulating lung neutrophil margination. Collectively, our data provides valuable insights into the fundamental regulation of neutrophil homeostasis, which may lead to the development of improved treatment regimens for neutropenic patients.

The cover of this issue is based on microscopy images of immature dendritic cells (DCs) stained with hematoxylin-and-eosin (H&E) provided by Chong et al. (pp. 1639–1651). In their article, Chong et al. investigate which cell types contribute to the differentiation of TNF/iNOS-producing dendritic cells (Tip-DCs), a subset of DCs that has been shown to arise during inflammation and to be an important mediator of immune defense. The authors provide evidence that CD8+ T cells can induce the rapid development of human monocytes into Tip-DCs that are capable of further driving Th1 responses by priming naive CD4+ T cells.

TNF/iNOS-producing dendritic cells (Tip-DCs) have been shown to arise during inflammation and are important mediators of immune defense. However, it is still relatively unclear which cell types contribute to their differentiation. Here we show that CD8(+) T cells, through the interaction with DCs, can induce the rapid development of human monocytes into Tip-DCs that express high levels of TNF-α and iNOS. Tip-DCs exhibited T-cell priming ability, expressed high levels of MHC class II, upregulated co-stimulatory molecules CD40, CD80, CD86, toll-like receptors TLR2, TLR3, TLR4, chemokine receptors CCR1 and CX3CR1 and expressed the classical mature DC marker, CD83. Differentiation of monocytes into Tip-DCs was partially dependent on IFN-γ as blocking the IFN-γ receptor on monocytes resulted in a significant decrease in CD40 and CD83 expression and in TNF-α production. Importantly, these Tip-DCs were capable of further driving Th1 responses by priming naive CD4(+) T cells for proliferation and IFN-γ production and this was partially dependent on Tip-DC production of TNF-α and NO. Our study hence identifies a role for CD8(+) T cells in orchestrating Th1-mediating signals through the differentiation of monocytes into Th1-inducing Tip-DCs.

TNF/iNOS-producing (Tip) dendritic cells have been shown to arise during inflammation and are important mediators of immune defense. However, it is still relatively unclear which cell types contribute to their differentiation. Here we show that CD8 T-cells, through the interaction with DCs, can induce the rapid development of human monocytes into Tip-DCs which express high levels of TNF-α and iNOS. These cells exhibited T-cell priming ability, expressed high levels of MHC class II, up-regulated co-stimulatory molecules CD40, CD80, CD86, toll-like receptors TLR2, TLR3, TLR4, chemokine receptors CCR1 and CX3CR1 and expressed the classical mature DC marker, CD83. Differentiation of monocytes into Tip-DCs was partially dependent on IFN-γ as blocking the IFN-γ receptor on monocytes resulted in a significant decrease in CD40, CD83 expression and TNF-α production. Importantly, these Tip-DCs were capable of further driving Th1 responses by priming naive CD4 T-cells for proliferation and IFN-γ production and this was partially dependent on Tip-DC’s production of TNF-α and NO. Our study hence identifies a role for CD8 T-cells in orchestrating Th1-mediating signals through the differentiation of monocytes into Th1-inducing Tip-DCs.