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Dendritic cells (DCs) and monocytes capture, transport, and present antigen to cognate T cells in the draining lymph nodes (LNs) in a CCR7-dependent manner. Since only migratory DCs express this chemokine receptor, it is unclear how monocytes reach the LN. In steady-state and following inhalation of several PAMPs, scRNA-seq identified LN mononuclear phagocytes as monocytes, resident, or migratory type 1 and type 2 conventional (c)DCs, despite the downregulation of Xcr1, Clec9a, H2-Ab1, Sirpa, and Clec10a transcripts on migratory cDCs. Migratory cDCs, however, upregulated Ccr7, Ccl17, Ccl22, and Ccl5. Migratory monocytes expressed Ccr5, a high-affinity receptor for Ccl5. Using two tracking methods, we observed that both CD88hiCD26lomonocytes and CD88-CD26hi cDCs captured inhaled antigens in the lung and migrated to LNs. Antigen exposure in mixed-chimeric Ccl5-, Ccr2-, Ccr5-, Ccr7-, and Batf3-deficient mice demonstrated that while antigen-bearing DCs use CCR7 to reach the LN, monocytes use CCR5 to follow CCL5-secreting migratory cDCs into the LN, where they regulate DC-mediated immunity., (© 2023 Rawat et al.)

Every immune response has accelerators and brakes. Depending on the pathogen or injury, monocytes can play either role, promoting or resolving immunity. Poly I:C, a potent TLR3 ligand, licenses cross-presenting dendritic cells (DC1) to accelerate a robust cytotoxic T cells response against a foreign antigen. Poly I:C thus has promise as an adjuvant in cancer immunotherapy and viral subunit vaccines. Like DC1s, monocytes are also abundant in the LNs. They may act as either immune accelerators or brakes, depending on the inflammatory mediator they encounter. However, little is known about their contribution to adaptive immunity in the context of antigen and Poly I:C. Using monocyte-deficient and chimeric mice, we demonstrate that LN monocytes indirectly dampen a Poly I:C induced antigen-specific cytotoxic T cell response, exerting a "braking" function. This effect is mediated by IL-10 production and induction of suppressor CD4 + T cells. In a metastatic melanoma model, we show that a triple-combination prophylactic treatment consisting of anti-IL-10, tumor peptides and Poly I:C works because removing IL-10 counteracts the monocytic brake, resulting in significantly fewer tumors compared to mice treated with tumor peptides and Poly I:C alone. Finally, in human LN tissue, we observed that monocytes (unlike DCs) express high levels of IL-10, suggesting that anti-IL-10 may be an important addition to treatments. Overall, our data demonstrates that LN monocytes regulate the induction of a robust DC1-mediated immune response. Neutralization of either IL-10 or monocytes can augment Poly I:C-based treatments and enhance T cell cytotoxicity., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Tewari, Prabagar, Gibbings, Rawat and Jakubzick.)

Interstitial macrophages (IMs) are present in multiple organs. Although there is limited knowledge of the unique functional role IM subtypes play, macrophages, in general, are known for their contribution in homeostatic tissue maintenance and inflammation such as clearing pathogens and debris and secreting inflammatory mediators and growth factors. IM subtypes have been identified in the heart, skin, and gut, and more recently we identified three distinct IMs in the lung. IMs express on their surface high levels of MerTK, CD64, and CD11b, with differences in CD11c, CD206, and MHC II expression, and referred to the three pulmonary IM subtypes as IM1 (CD11c lo CD206 + MHCII lo ), IM2 (CD11c lo CD206 + MHCII hi ), and IM3 (CD11c hi CD206 lo MHCII hi ). In this chapter, we highlight how to extract IMs from the lung using three different digestion enzymes: elastase, collagenase D, and Liberase TM. Of these three commonly used enzymes, Liberase TM was the most effective at IM extraction, particularly IM3. Furthermore, alternative staining strategies to identify IMs were examined, which included CD64, MerTK, F4/80, and Tim4. Thus, future studies highlighting the functional role of IM subtypes will help further our understanding of how tissue homeostasis is maintained and inflammatory conditions are induced and resolved.

Mononuclear phagocytes (MP) consist of macrophages, dendritic cells (DCs), and monocytes. In all organs, including the lung, there are multiple subtypes within these categories. The existence of all these cell types suggest that there is a clear division of labor and delicate balance between the MPs under steady state and inflammatory conditions. Although great strides have been made to understand MPs in the mouse lung, and human blood, little is known about the MPs that exist in the human lung and lung-draining lymph nodes (LNs), and even less is known about their functional roles, studies of which will require a large number of sorted cells. We have comprehensively examined cell surface markers previously used in a variety of organs to identify human pulmonary MPs. In the lung, we consistently identify five extravascular pulmonary MPs and three LN MPs. These MPs were present in over 100 lungs regardless of age or gender. Notably, the human blood CD141 + DCs, as described in the literature, were not observed in non-diseased lungs or their draining LNs. In the lung and draining LNs, expression of CD141 was only observed on HLADR + CD11c + CD14 + extravascular monocytes (often confused in the LN as resident DCs based on the level of HLADR expression and mouse LN data). In the human lung and LNs there are at least two DC subtypes expressing HLADR, DEC205 and CD1c, along with circulating monocytes that behave as either antigen-presenting cells or macrophages. Furthermore, we demonstrate how to distinguish between alveolar macrophages and interstitial macrophage subtypes. It still remains unclear how the human pulmonary MPs identified here align with mouse MPs. Clearly, we are now past the stage of cell surface marker characterization, and future studies will need to move toward understanding what these cell types are and how they function. Our hope is that the strategy described here can help the pulmonary community take this next step.

Recently it was shown that circulating Ly6C(+) monocytes traffic from tissue to the draining lymph nodes (LNs) with minimal alteration in their overall phenotype. Furthermore, in the steady state, Ly6C(+) monocytes are as abundant as classical dendritic cells (DCs) within the draining LNs, and even more abundant during inflammation. However, little is known about the functional roles of constitutively trafficking Ly6C(+) monocytes. In this study we investigated whether Ly6C(+) monocytes can efferocytose (acquire dying cells) and cross-present cell-associated antigen, a functional property particularly attributed to Batf3(+) DCs. We demonstrated that Ly6C(+) monocytes intrinsically efferocytose and cross-present cell-associated antigen to CD8(+) T cells. In addition, efferocytosis was enhanced upon direct activation of the Ly6C(+) monocytes through its corresponding TLRs, TLR4 and TLR7. However, only ligation of TLR7, and not TLR4, enhanced cross-presentation by Ly6C(+) monocytes. Overall, this study outlines two functional roles, among others, that Ly6C(+) monocytes have during an adaptive immune response.

It is thought that monocytes rapidly differentiate to macrophages or dendritic cells (DCs) upon leaving blood. Here we have shown that Ly-6C⁺ monocytes constitutively trafficked into skin, lung, and lymph nodes (LNs). Entry was unaffected in gnotobiotic mice. Monocytes in resting lung and LN had similar gene expression profiles to blood monocytes but elevated transcripts of a limited number of genes including cyclo-oxygenase-2 (COX-2) and major histocompatibility complex class II (MHCII), induced by monocyte interaction with endothelium. Parabiosis, bromodoxyuridine (BrdU) pulse-chase analysis, and intranasal instillation of tracers indicated that instead of contributing to resident macrophages in the lung, recruited endogenous monocytes acquired antigen for carriage to draining LNs, a function redundant with DCs though differentiation to DCs did not occur. Thus, monocytes can enter steady-state nonlymphoid organs and recirculate to LNs without differentiation to macrophages or DCs, revising a long-held view that monocytes become tissue-resident macrophages by default., (Copyright © 2013 Elsevier Inc. All rights reserved.)