facial-vein-venipuncture-for-murine-blood-collection

Facial Vein Venipuncture for Murine Blood Collection

This paper presents an optimized protocol for murine blood collection via facial vein venipuncture using a lancet, offering a rapid, minimally invasive, ...

Dartmouth Geisel School of Medicine

The role of recruitment versus training in influenza-induced lasting changes to alveolar macrophage function.

Myeloid, T, and NK cells are key players in the elimination phase of cancer immunoediting, also referred to as cancer immunosurveillance. However, the role of B cells and NAbs, which are present prior to the encounter with cognate antigens, has been overlooked. One reason is due to the popular use of a single B cell-deficient mouse model, muMT mice. Cancer models using muMT mice display a similar tumor burden as their wild-type (WT) counterparts. Empirically, we observe what others have previously reported with muMT mice. However, using two other B cell-deficient mouse models (IgHELMD4 and CD19), we show a three- to fivefold increase in tumor burden relative to WT mice. In addition, using an unconventional, non-cancer-related, immune neoantigen model where hypoxic conditions and cell clustering are absent, we provide evidence that B cells and their innate, natural antibodies (NAbs) are critical for the detection and elimination of neoantigen-expressing cells. Finally, we find that muMT mice display anti-tumor immunity because of an unexpected compensatory mechanism consisting of significantly enhanced type 1 interferon (IFN)-producing plasmacytoid dendritic cells (pDCs), which recruit a substantial number of NK cells to the tumor microenvironment compared to WT mice. Diminishing this compensatory pDC-IFN-NK cell mechanism revealed that muMT mice develop a three- to fivefold increase in tumor burden compared to WT mice. In summary, our findings suggest that NAbs are part of an early defense against not only microorganisms and dying cells, but precancerous cells as well.

Redefining innate natural antibodies as important contributors to anti-tumor immunity.

Alveolar macrophages (AMs) reside on the luminal surface of the airways and alveoli, ensuring proper gas exchange by ingesting cellular debris and pathogens, and regulating inflammatory responses. Therefore, understanding the heterogeneity and diverse roles played by AMs, interstitial macrophages, and recruited monocytes is critical for treating airway diseases. We performed single-cell RNA sequencing on 113,213 bronchoalveolar lavage cells from four healthy and three uninflamed cystic fibrosis subjects and identified two MARCKSLGMNIMs, FOLR2SELENOP and SPP1PLA2G7 IMs, monocyte subtypes, DC1, DC2, migDCs, plasmacytoid DCs, lymphocytes, epithelial cells, and four AM superclusters (families) based on the gene expression of  and  These four AM families have at least eight distinct functional members (subclusters) named after their differentially expressed gene(s): IGF1, CCL18, CXCL5, cholesterol, chemokine, metallothionein, interferon, and small-cluster AMs. Interestingly, the chemokine cluster further divides with each subcluster selectively expressing a unique combination of chemokines. One of the most striking observations, besides the heterogeneity, is the conservation of AM family members in relatively equal ratio across all AM superclusters and individuals. Transcriptional data and TotalSeq technology were used to investigate cell surface markers that distinguish resident AMs from recruited monocytes. Last, other AM datasets were projected onto our dataset. Similar AM superclusters and functional subclusters were observed, along with a significant increase in chemokine and IFN AM subclusters in individuals infected with COVID-19. Overall, functional specializations of the AM subclusters suggest that there are highly regulated AM niches with defined programming states, highlighting a clear division of labor.

ScRNA-seq expression of  and  identifies four alveolar macrophage superclusters in healthy BALF.

Recent studies have revealed a critical role for natural Abs (NAbs) in antitumor immune responses. However, the role of NAbs in cancer immunosurveillance remains unexplored, mainly because of the lack of in vivo models that mimic the early recognition and elimination of transforming cells. In this article, we propose a role for NAbs in alerting the immune system against precancerous neoantigen-expressing cells immediately after they escape intrinsic tumor suppression mechanisms. We identify four distinct reproducible, trackable, MHC-matched neoantigen-expressing cell models that do not form tumors as the end point. This amplified readout in the critical window prior to tumor formation allows investigation of new mediators of cancer immunosurveillance. We found that neoantigen-expressing cells adoptively transferred in NAb-deficient mice persisted, whereas they were eliminated in wild-type mice, indicating that the circulating NAb repertoire alerts the immune system to the presence of transformed cells. Moreover, immunity is mounted against immunogenic and nonimmunogenic neoantigens contained in the NAb-tagged cells, regardless of whether the NAb directly recognizes the neoantigens. Beyond these neoantigen-expressing model systems, we observed a significantly greater tumor burden in chemically and virally induced tumor models in NAb-deficient mice compared with wild-type mice. Restoration of the NAb repertoire in NAb-deficient mice elicited the recognition and elimination of neoantigen-expressing cells and cancer. These data show that NAbs are required and sufficient for elimination of transformed cells early in tumorigenesis. These models can now be used to investigate how NAbs stimulate immunity via recognition receptors to eliminate precancerous cells.

Natural Antibodies Alert the Adaptive Immune System of the Presence of Transformed Cells in Early Tumorigenesis.

Tissue-resident alveolar and interstitial macrophages and recruited macrophages are critical players in innate immunity and maintenance of lung homeostasis. Until recently, assessing the differential functional contributions of tissue-resident versus recruited macrophages has been challenging because they share overlapping cell surface markers, making it difficult to separate them using conventional methods. This review describes how scRNA-seq and spatial transcriptomics can separate these subpopulations and help unravel the complexity of macrophage biology in homeostasis and disease. First, we provide a guide to identifying and distinguishing lung macrophages from other mononuclear phagocytes in humans and mice. Second, we outline emerging concepts related to the development and function of the various lung macrophages in the alveolar, perivascular, and interstitial niches. Finally, we describe how different tissue states profoundly alter their functions, including acute and chronic lung disease, cancer, and aging.

Biology of lung macrophages in health and disease.

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.

CCL5-producing migratory dendritic cells guide CCR5+ monocytes into the draining lymph nodes.

Monocytes can differentiate into tissue-resident pleural macrophages, but the mechanisms underlying this process are not yet fully understood. In this issue of Immunity, Finlay et al. show that Th2 cytokines promote this differentiation in resistant mice infected with Litomosoides sigmodontis.

Macrophages show up in style when Th2 lymphocytes organize their homecoming.

Transplantation and cancer expose the immune system to neoantigens, including immunogenic (dominant and subdominant) and nonimmunogenic Ags with varying quantities and affinities of immunodominant peptides. Conceptually, immunity is believed to mainly target dominant Ags when subdominant or nondominant Ags are linked within the same cell due to T cell interference. This phenomenon is called immunodominance. However, our previous study in mice showed that linked nonimmunogenic Ags (OVA and GFP) containing immunodominant peptides mount immunity irrespective of the MHC-matched allogeneic cell's immunogenicity. Consequently, we further explored 1) under what circumstances does the congenic marker CD45.1 provoke immunity in CD45.2 mice, and 2) whether linking two dominant or subdominant Ags can instigate an immune response. Our observations showed that CD45.1 (or CD45.2), when connected to low-immunogenic cell types is presented as an immunogen, which contrasts with its outcome when linked to high-immunogenic cell types. Moreover, we found that both dominant and subdominant Ags are presented as immunogens when linked in environments with lower immunogenic thresholds. These findings challenge the existing perception that immunity is predominantly elicited against dominant Ags when linked to subdominant or nondominant Ags. This study takes a fundamental step toward understanding the nuanced relationship between immunogenic and nonimmunogenic Ags, potentially opening new avenues for comprehending cancer immunoediting and enhancing the conversion of cold tumors with low immunogenicity into responsive hot tumors.

Immunogenicity Threshold in Allogeneic Cells Impacts CTL Response to Nondominant Congenic Antigens.

Lung-resident macrophages, which include alveolar macrophages and interstitial macrophages (IMs), exhibit a high degree of diversity, generally attributed to different activation states, and often complicated by the influx of monocytes into the pool of tissue-resident macrophages. To gain a deeper insight into the functional diversity of IMs, here we perform comprehensive transcriptional profiling of resident IMs and reveal ten distinct chemokine-expressing IM subsets at steady state and during inflammation. Similar IM subsets that exhibited coordinated chemokine signatures and differentially expressed genes were observed across various tissues and species, indicating conserved specialized functional roles. Other macrophage types shared specific IM chemokine profiles, while also presenting their own unique chemokine signatures. Depletion of CD206 IMs in Pf4R26 and Pf4R26Cx3cr1 mice led to diminished inflammatory cell recruitment, reduced tertiary lymphoid structure formation and fewer germinal center B cells in models of allergen- and infection-driven inflammation. These observations highlight the specialized roles of IMs, defined by their coordinated chemokine production, in regulating immune cell influx and organizing tertiary lymphoid tissue architecture.

Coordinated chemokine expression defines macrophage subsets across tissues.

The Heterogeneity, Parallels, and Divergence of Alveolar Macrophages in Humans and Mice.

In this Perspective, we review the major themes from a Basic Science Core session at ATS 2024: Multi-omic and single-cell approaches for elucidating cellular biology and pathobiology of lung disease, with a focus on novel biology identified in human lungs. Recent advances have enabled researchers to study nucleic acids, proteins, and metabolites at single-cell resolution in the lung. These tools can be integrated with complementary methods to lead to new discoveries about lung function and develop treatments for respiratory diseases. We discuss the role of multicenter consortia in developing lung cell atlases and highlight the role of single-cell methods in identifying new respiratory cell types. We then review advances in several respiratory diseases that demonstrate the use of these tools to study lung development, injury, and repair. Lastly, we consider future directions for the field, including efforts to harmonize cell types across studies and to develop more robust analytical pipelines.

Article	Year
The role of recruitment versus training in influenza-induced lasting changes to alveolar macrophage function. Nature immunology\| PubMed ID: 37640788	2023
Redefining innate natural antibodies as important contributors to anti-tumor immunity. eLife\| PubMed ID: 34608861	2021
ScRNA-seq expression of and identifies four alveolar macrophage superclusters in healthy BALF. Life science alliance\| PubMed ID: 35820705	2022
Natural Antibodies Alert the Adaptive Immune System of the Presence of Transformed Cells in Early Tumorigenesis. Journal of immunology (Baltimore, Md. : 1950)\| PubMed ID: 36028292	2022
Biology of lung macrophages in health and disease. Immunity\| PubMed ID: 36103853	2022
CCL5-producing migratory dendritic cells guide CCR5+ monocytes into the draining lymph nodes. The Journal of experimental medicine\| PubMed ID: 36946983	2023
Macrophages show up in style when Th2 lymphocytes organize their homecoming. Immunity\| PubMed ID: 37163989	2023
Immunogenicity Threshold in Allogeneic Cells Impacts CTL Response to Nondominant Congenic Antigens. Journal of immunology (Baltimore, Md. : 1950)\| PubMed ID: 37850969	2023
Coordinated chemokine expression defines macrophage subsets across tissues. Nature immunology\| PubMed ID: 38698086	2024
The Heterogeneity, Parallels, and Divergence of Alveolar Macrophages in Humans and Mice. American journal of respiratory cell and molecular biology\| PubMed ID: 39535274	2025
Multi-omic and Single-Cell Approaches for Elucidating Cell Biology and Pathobiology of Lung Disease. American journal of respiratory cell and molecular biology\| PubMed ID: 40700713	2025

Claudia V. Jakubzick

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