Non-mutational neoantigens in disease.

The ability of mammals to mount adaptive immune responses culminating with the establishment of immunological memory is predicated on the ability of the mature T cell repertoire to recognize antigenic peptides presented by syngeneic MHC class I and II molecules. Although it is widely believed that mature T cells are highly skewed towards the recognition of antigenic peptides originating from genetically diverse (for example, foreign or mutated) protein-coding regions, preclinical and clinical data rather demonstrate that novel antigenic determinants efficiently recognized by mature T cells can emerge from a variety of non-mutational mechanisms. In this Review, we describe various mechanisms that underlie the formation of bona fide non-mutational neoantigens, such as epitope mimicry, upregulation of cryptic epitopes, usage of non-canonical initiation codons, alternative RNA splicing, and defective ribosomal RNA processing, as well as both enzymatic and non-enzymatic post-translational protein modifications. Moreover, we discuss the implications of the immune recognition of non-mutational neoantigens for human disease.

Chaperone-mediated autophagy prevents collapse of the neuronal metastable proteome.

Components of the proteostasis network malfunction in aging, and reduced protein quality control in neurons has been proposed to promote neurodegeneration. Here, we investigate the role of chaperone-mediated autophagy (CMA), a selective autophagy shown to degrade neurodegeneration-related proteins, in neuronal proteostasis. Using mouse models with systemic and neuronal-specific CMA blockage, we demonstrate that loss of neuronal CMA leads to altered neuronal function, selective changes in the neuronal metastable proteome, and proteotoxicity, all reminiscent of brain aging. Imposing CMA loss on a mouse model of Alzheimer's disease (AD) has synergistic negative effects on the proteome at risk of aggregation, thus increasing neuronal disease vulnerability and accelerating disease progression. Conversely, chemical enhancement of CMA ameliorates pathology in two different AD experimental mouse models. We conclude that functional CMA is essential for neuronal proteostasis through the maintenance of a subset of the proteome with a higher risk of misfolding than the general proteome.

Pleiotropic consequences of metabolic stress for the major histocompatibility complex class II molecule antigen processing and presentation machinery.

Hyperglycemia and hyperlipidemia are often observed in individuals with type II diabetes (T2D) and related mouse models. One dysmetabolic biochemical consequence is the non-enzymatic reaction between sugars, lipids, and proteins, favoring protein glycation, glycoxidation, and lipoxidation. Here, we identified oxidative alterations in key components of the major histocompatibility complex (MHC) class II molecule antigen processing and presentation machinery in vivo under conditions of hyperglycemia-induced metabolic stress. These modifications were linked to epitope-specific changes in endosomal processing efficiency, MHC class II-peptide binding, and DM editing activity. Moreover, we observed some quantitative and qualitative changes in the MHC class II immunopeptidome of Ob/Ob mice on a high-fat diet compared with controls, including changes in the presentation of an apolipoprotein B100 peptide associated previously with T2D and metabolic syndrome-related clinical complications. These findings highlight a link between glycation reactions and altered MHC class II antigen presentation that may contribute to T2D complications.

A Relay Pathway between Arginine and Tryptophan Metabolism Confers Immunosuppressive Properties on Dendritic Cells.

Arginase 1 (Arg1) and indoleamine 2,3-dioxygenase 1 (IDO1) are immunoregulatory enzymes catalyzing the degradation of l-arginine and l-tryptophan, respectively, resulting in local amino acid deprivation. In addition, unlike Arg1, IDO1 is also endowed with non-enzymatic signaling activity in dendritic cells (DCs). Despite considerable knowledge of their individual biology, no integrated functions of Arg1 and IDO1 have been reported yet. We found that IDO1 phosphorylation and consequent activation of IDO1 signaling in DCs was strictly dependent on prior expression of Arg1 and Arg1-dependent production of polyamines. Polyamines, either produced by DCs or released by bystander Arg1+ myeloid-derived suppressor cells, conditioned DCs toward an IDO1-dependent, immunosuppressive phenotype via activation of the Src kinase, which has IDO1-phosphorylating activity. Thus our data indicate that Arg1 and IDO1 are linked by an entwined pathway in immunometabolism and that their joint modulation could represent an important target for effective immunotherapy in several disease settings.

Autophagy in major human diseases.

Autophagy is a core molecular pathway for the preservation of cellular and organismal homeostasis. Pharmacological and genetic interventions impairing autophagy responses promote or aggravate disease in a plethora of experimental models. Consistently, mutations in autophagy-related processes cause severe human pathologies. Here, we review and discuss preclinical data linking autophagy dysfunction to the pathogenesis of major human disorders including cancer as well as cardiovascular, neurodegenerative, metabolic, pulmonary, renal, infectious, musculoskeletal, and ocular disorders.

Pancreatic islets communicate with lymphoid tissues via exocytosis of insulin peptides.

Tissue-specific autoimmunity occurs when selected antigens presented by susceptible alleles of the major histocompatibility complex are recognized by T cells. However, the reason why certain specific self-antigens dominate the response and are indispensable for triggering autoreactivity is unclear. Spontaneous presentation of insulin is essential for initiating autoimmune type 1 diabetes in non-obese diabetic mice1,2. A major set of pathogenic CD4 T cells specifically recognizes the 12-20 segment of the insulin B-chain (B:12-20), an epitope that is generated from direct presentation of insulin peptides by antigen-presenting cells3,4. These T cells do not respond to antigen-presenting cells that have taken up insulin that, after processing, leads to presentation of a different segment representing a one-residue shift, B:13-214. CD4 T cells that recognize B:12-20 escape negative selection in the thymus and cause diabetes, whereas those that recognize B:13-21 have only a minor role in autoimmunity3-5. Although presentation of B:12-20 is evident in the islets3,6, insulin-specific germinal centres can be formed in various lymphoid tissues, suggesting that insulin presentation is widespread7,8. Here we use live imaging to document the distribution of insulin recognition by CD4 T cells throughout various lymph nodes. Furthermore, we identify catabolized insulin peptide fragments containing defined pathogenic epitopes in ß-cell granules from mice and humans. Upon glucose challenge, these fragments are released into the circulation and are recognized by CD4 T cells, leading to an activation state that results in transcriptional reprogramming and enhanced diabetogenicity. Therefore, a tissue such as pancreatic islets, by releasing catabolized products, imposes a constant threat to self-tolerance. These findings reveal a self-recognition pathway underlying a primary autoantigen and provide a foundation for assessing antigenic targets that precipitate pathogenic outcomes by systemically sensitizing lymphoid tissues.

Positive allosteric modulation of indoleamine 2,3-dioxygenase 1 restrains neuroinflammation.

l-tryptophan (Trp), an essential amino acid for mammals, is the precursor of a wide array of immunomodulatory metabolites produced by the kynurenine and serotonin pathways. The kynurenine pathway is a paramount source of several immunoregulatory metabolites, including l-kynurenine (Kyn), the main product of indoleamine 2,3-dioxygenase 1 (IDO1) that catalyzes the rate-limiting step of the pathway. In the serotonin pathway, the metabolite N-acetylserotonin (NAS) has been shown to possess antioxidant, antiinflammatory, and neuroprotective properties in experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis (MS). However, little is known about the exact mode of action of the serotonin metabolite and the possible interplay between the 2 Trp metabolic pathways. Prompted by the discovery that NAS neuroprotective effects in EAE are abrogated in mice lacking IDO1 expression, we investigated the NAS mode of action in neuroinflammation. We found that NAS directly binds IDO1 and acts as a positive allosteric modulator (PAM) of the IDO1 enzyme in vitro and in vivo. As a result, increased Kyn will activate the ligand-activated transcription factor aryl hydrocarbon receptor and, consequently, antiinflammatory and immunoregulatory effects. Because NAS also increased IDO1 activity in peripheral blood mononuclear cells of a significant proportion of MS patients, our data may set the basis for the development of IDO1 PAMs as first-in-class drugs in autoimmune/neuroinflammatory diseases.

Advances in understanding and examining lymphatic function: relevance for understanding autoimmunity.

PURPOSE OF REVIEW: The aim of this review is to give insights into how novel lymphatics functions may influence autoimmunity. RECENT FINDINGS: The lymphatic system connects peripheral tissues to draining lymph nodes to regulate adaptive immunity and directly interfaces with leukocytes in lymph vessels and in the lymph node. Here, we discuss recent findings showing evidence of dysfunctional lymphatics in autoimmune disease, new understanding of how afferent lymphatic regulation can modulate immunity, lymph node lymphatic heterogeneity and how these lymphatics can directly modulate lymphocyte function, how this understanding can be harnessed for new therapeutics, and new tools for the investigation of lymphatic and immune biology. SUMMARY: Lymphatics have an active role in the regulation of inflammation and the adaptive immune response. Here, we review recent findings in lymphatics biology in peripheral tissues and lymph nodes and emphasize the relevance for better understanding autoimmune diseases.

Class IA PI3Ks regulate subcellular and functional dynamics of IDO1.

Knowledge of a protein's spatial dynamics at the subcellular level is key to understanding its function(s), interactions, and associated intracellular events. Indoleamine 2,3-dioxygenase 1 (IDO1) is a cytosolic enzyme that controls immune responses via tryptophan metabolism, mainly through its enzymic activity. When phosphorylated, however, IDO1 acts as a signaling molecule in plasmacytoid dendritic cells (pDCs), thus activating genomic effects, ultimately leading to long-lasting immunosuppression. Whether the two activities-namely, the catalytic and signaling functions-are spatially segregated has been unclear. We found that, under conditions favoring signaling rather than catabolic events, IDO1 shifts from the cytosol to early endosomes. The event requires interaction with class IA phosphoinositide 3-kinases (PI3Ks), which become activated, resulting in full expression of the immunoregulatory phenotype in vivo in pDCs as resulting from IDO1-dependent signaling events. Thus, IDO1's spatial dynamics meet the needs for short-acting as well as durable mechanisms of immune suppression, both under acute and chronic inflammatory conditions. These data expand the theoretical basis for an IDO1-centered therapy in inflammation and autoimmunity.

Molecular definitions of autophagy and related processes.

Over the past two decades, the molecular machinery that underlies autophagic responses has been characterized with ever increasing precision in multiple model organisms. Moreover, it has become clear that autophagy and autophagy-related processes have profound implications for human pathophysiology. However, considerable confusion persists about the use of appropriate terms to indicate specific types of autophagy and some components of the autophagy machinery, which may have detrimental effects on the expansion of the field. Driven by the overt recognition of such a potential obstacle, a panel of leading experts in the field attempts here to define several autophagy-related terms based on specific biochemical features. The ultimate objective of this collaborative exchange is to formulate recommendations that facilitate the dissemination of knowledge within and outside the field of autophagy research.

Message in a vesicle - trans-kingdom intercommunication at the vector-host interface.

Vector-borne diseases cause over 700,000 deaths annually and represent 17% of all infectious illnesses worldwide. This public health menace highlights the importance of understanding how arthropod vectors, microbes and their mammalian hosts interact. Currently, an emphasis of the scientific enterprise is at the vector-host interface where human pathogens are acquired and transmitted. At this spatial junction, arthropod effector molecules are secreted, enabling microbial pathogenesis and disease. Extracellular vesicles manipulate signaling networks by carrying proteins, lipids, carbohydrates and regulatory nucleic acids. Therefore, they are well positioned to aid in cell-to-cell communication and mediate molecular interactions. This Review briefly discusses exosome and microvesicle biogenesis, their cargo, and the role that nanovesicles play during pathogen spread, host colonization and disease pathogenesis. We then focus on the role of extracellular vesicles in dictating microbial pathogenesis and host immunity during transmission of vector-borne pathogens.

Lymphatic Cannulation for Lymph Sampling and Molecular Delivery.

Unlike the blood, the interstitial fluid and the deriving lymph are directly bathing the cellular layer of each organ. As such, composition analysis of the lymphatic fluid can provide more precise biochemical and cellular information on an organ's health and be a valuable resource for biomarker discovery. In this study, we describe a protocol for cannulation of mouse and rat lymphatic collectors that is suitable for the following: the "omic" sampling of pre- and postnodal lymph, collected from different anatomical districts; the phenotyping of immune cells circulating between parenchymal organs and draining lymph nodes; injection of known amounts of molecules for quantitative immunological studies of nodal trafficking and/or clearance; and monitoring an organ's biochemical omic changes in pathological conditions. Our data indicate that probing the lymphatic fluid can provide an accurate snapshot of an organ's physiology/pathology, making it an ideal target for liquid biopsy.

Contribution of the plasma and lymph Degradome and Peptidome to the MHC Ligandome.

Every biological fluid, blood, interstitial fluid and lymph, urine, saliva, lacrimal fluid, nipple aspirate, and spinal fluid, contains a peptidome-degradome derived from the cellular secretome along with byproducts of the metabolic/catabolic activities of each parenchymal organ. Clement et al. (J Proteomics 78:172-187, 2013), Clement et al. (J Biol Chem 291:5576-5595, 2016), Clement et al. (PLoS One 5:e9863, 2010), Clement et al. (Trends Immunol 32:6-11, 2011), Clement et al. (Front Immunol 4:424, 2013), Geho et al. (Curr Opin Chem Biol 10, 50-55, 2006), Interewicz et al. (Lymphology 37:65­72, 2004), Leak et al. (Proteomics 4:753­765, 2004), Popova et al. (PLoS One 9:e110873, 2014), Zhou et al. (Electrophoresis 25:1289­1298, 2004), D'Alessandro et al. (Shock 42:509­517, 2014), Dzieciatkowska et al. (Shock 42:485­498, 2014), Dzieciatkowska et al. (Shock 35:331­338, 2011), Jordan et al. (J Surg Res 143:130­135, 2007), Peltz et al. (Surgery 146:347­357, 2009), Zurawel et al. (Clin Proteomics 8:1, 2011), Ling et al. (Clin Proteomics 6:175­193, 2010), Sturm et al. (Nat Commun 4:1616, 2013). Over the last decade, qualitative and quantitative analysis of the biological fluids peptidome and degradome have provided a dynamic measurement of tissue homeostasis as well as the tissue response to pathological damage. Proteomic profiling has mapped several of the proteases and resulting degradation by-products derived from cell cycle progression, organ/tissue remodeling and cellular growth, physiological apoptosis, hemostasis, and angiogenesis. Currently, a growing interest lies in the degradome observed during pathological conditions such as cancer, autoimmune diseases, and immune responses to pathogens as a way to exploit biological fluids as liquid biopsies for biomarker discovery Dzieciatkowska et al. (Shock 42:485-498, 2014), Dzieciatkowska et al. (Shock 35:331-338, 2011), Ling et al. (Clin Proteomics 6:175-193, 2010), Ugalde et al. (Methods Mol Biol 622:3-29, 2010), Quesada et al. (Nucleic Acids Res 37:D239­243, 2009), Cal et al. (Front Biosci 12, 4661-4669, 2007), Shen et al. (PLoS One 5:e13133, 2010a), Antwi et al. (Mol Immunol 46:2931-2937, 2009a), Antwi et al. (J Proteome Res 8:4722­4731, 2009b), Bedin et al. (J Cell Physiol 231, 915­925, 2016), Bery et al. (Clin Proteomics 11:13, 2014), Bhalla et al. (Sci Rep 7:1511, 2017), Fan et al. (Diagn Pathol 7:45, 2012a), Fang et al. (Shock 34:291­298, 2010), Fiedler et al. (Clin Cancer Res 15:3812­3819, 2009), Fredolini et al. (AAPS J 12:504­518, 2010), Greening et al. (Enzymes 42:27­64, 2017), He et al. (PLoS One 8:e63724, 2013), Huang et al. (Int J Gynecol Cancer 28:355­362, 2018), Hashiguchi et al. (Med Hypotheses 73:760­763, 2009), Liotta and Petricoin (J Clin Invest 116:26­30, 2006), Petricoin et al. (Nat Rev Cancer 6:961­967, 2006), Shen et al. (J Proteome Res 9:2339­2346, 2010a), Shen et al. (J Proteome Res 5:3154­3160, 2006), Smith (Clin Proteomics 11:23, 2014), Wang et al. (Oncotarget 8:59376­59386, 2017), Yang et al. (Clin Exp Med 12:79­87, 2012a), Yang et al. (J Clin Lab Anal 26:148­154, 2012b), Yang et al. (Anat Rec (Hoboken) 293:2027­2033, 2010), Zapico-Muniz et al. (Pancreas 39:1293­1298, 2010), Villanueva et al. (Mol Cell Proteomics 5:1840­1852, 2006), Robbins et al. (J Clin Oncol 23:4835­4837, 2005), Klupczynska et al. (Int J Mol Sci 17:410, 2016). In this review, we focus on the current knowledge of the degradome/peptidome observed in two main biological fluids (plasma and lymph) during physiological and pathological conditions and its importance for immune surveillance.

In vitro model reveals a role for mechanical stretch in the remodeling response of lymphatic muscle cells.

OBJECTIVE: Using primary LMCs in vitro, we sought to characterize the impact of LMC remodeling on their functional and molecular response to mechanical loading and culture conditions. METHODS: Primary "wounded leg" LMCs were derived from the hindlimb of three sheep who underwent lymphatic injury 6 weeks prior, while "control leg" LMCs were derived from the contralateral, unwounded, limb. Function of the LMCs was characterized in response to media of variable levels of serum (10% vs 0.2%) and glucose (4.5 vs 1 g/L). Functional and proteomic data were evaluated in LMCs exposed to cyclic stretch (0.1 Hz, 7.5% elongation) for 1 week. RESULTS: LMCs were sensitive to changes in serum levels, significantly reducing overall activity and collagen synthesis under low serum conditions. LMCs from the remodeled vessel had higher baseline levels of metabolic activity but not collagen synthesis. Cyclic loading induced cellular alignment perpendicular to the axis of stretch and alterations in signaling pathways associated with metabolism. Remodeled LMCs had consistently higher levels of metabolic activity and were more resistant to strain-induced apoptosis. CONCLUSIONS: LMCs exist on a functional spectrum, becoming more active in response to stretching and maintaining phenotypic remodeling in response to local lymphatic/tissue damage.

Patterns of expression of factor VIII and von Willebrand factor by endothelial cell subsets in vivo.

UNLABELLED: Circulating factor VIII (FVIII) is derived from liver and from extrahepatic sources probably of endothelial origin, but the vascular sites of FVIII production remain unclear. Among organs profiled, only liver and lymph nodes (LNs) show abundant expression of F8 messenger RNA (mRNA). Transcriptomic profiling of subsets of stromal cells, including endothelial cells (ECs) from mouse LNs and other tissues, showed that F8 mRNA is expressed by lymphatic ECs (LECs) but not by capillary ECs (capECs), fibroblastic reticular cells, or hematopoietic cells. Among blood ECs profiled, F8 expression was seen only in fenestrated ECs (liver sinusoidal and renal glomerular ECs) and some high endothelial venules. In contrast, von Willebrand factor mRNA was expressed in capECs but not in LECs; it was coexpressed with F8 mRNA in postcapillary high endothelial venules. Purified LECs and liver sinusoidal ECs but not capECs from LNs secrete active FVIII in culture, and human and mouse lymph contained substantial FVIII: C activity. Our results revealed localized vascular expression of FVIII and von Willebrand factor and identified LECs as a major cellular source of FVIII in extrahepatic tissues.

The Dendritic Cell Major Histocompatibility Complex II (MHC II) Peptidome Derives from a Variety of Processing Pathways and Includes Peptides with a Broad Spectrum of HLA-DM Sensitivity.

The repertoire of peptides displayed in vivo by MHC II molecules derives from a wide spectrum of proteins produced by different cell types. Although intracellular endosomal processing in dendritic cells and B cells has been characterized for a few antigens, the overall range of processing pathways responsible for generating the MHC II peptidome are currently unclear. To determine the contribution of non-endosomal processing pathways, we eluted and sequenced over 3000 HLA-DR1-bound peptides presented in vivo by dendritic cells. The processing enzymes were identified by reference to a database of experimentally determined cleavage sites and experimentally validated for four epitopes derived from complement 3, collagen II, thymosin ß4, and gelsolin. We determined that self-antigens processed by tissue-specific proteases, including complement, matrix metalloproteases, caspases, and granzymes, and carried by lymph, contribute significantly to the MHC II self-peptidome presented by conventional dendritic cells in vivo. Additionally, the presented peptides exhibited a wide spectrum of binding affinity and HLA-DM susceptibility. The results indicate that the HLA-DR1-restricted self-peptidome presented under physiological conditions derives from a variety of processing pathways. Non-endosomal processing enzymes add to the number of epitopes cleaved by cathepsins, altogether generating a wider peptide repertoire. Taken together with HLA-DM-dependent and-independent loading pathways, this ensures that a broad self-peptidome is presented by dendritic cells. This work brings attention to the role of "self-recognition" as a dynamic interaction between dendritic cells and the metabolic/catabolic activities ongoing in every parenchymal organ as part of tissue growth, remodeling, and physiological apoptosis.

Structural and Biological Interaction of hsc-70 Protein with Phosphatidylserine in Endosomal Microautophagy.

hsc-70 (HSPA8) is a cytosolic molecular chaperone, which plays a central role in cellular proteostasis, including quality control during protein refolding and regulation of protein degradation. hsc-70 is pivotal to the process of macroautophagy, chaperone-mediated autophagy, and endosomal microautophagy. The latter requires hsc-70 interaction with negatively charged phosphatidylserine (PS) at the endosomal limiting membrane. Herein, by combining plasmon resonance, NMR spectroscopy, and amino acid mutagenesis, we mapped the C terminus of the hsc-70 LID domain as the structural interface interacting with endosomal PS, and we estimated an hsc-70/PS equilibrium dissociation constant of 4.7 ± 0.1 µm. This interaction is specific and involves a total of 4-5 lysine residues. Plasmon resonance and NMR results were further experimentally validated by hsc-70 endosomal binding experiments and endosomal microautophagy assays. The discovery of this previously unknown contact surface for hsc-70 in this work elucidates the mechanism of hsc-70 PS/membrane interaction for cytosolic cargo internalization into endosomes.

The lipid kinase PI4KIIIß preserves lysosomal identity.

Lipid modifications are essential in cellular sorting and trafficking inside cells. The role of phosphoinositides in trafficking between Golgi and endocytic/lysosomal compartments has been extensively explored and the kinases responsible for these lipid changes have been identified. In contrast, the mechanisms that mediate exit and recycling from lysosomes (Lys), considered for a long time as terminal compartments, are less understood. In this work, we identify a dynamic association of the lipid kinase PI4KIIIß with Lys and unveil its regulatory function in lysosomal export and retrieval. We have found that absence of PI4KIIIß leads to abnormal formation of tubular structures from the lysosomal surface and loss of lysosomal constituents through these tubules. We demonstrate that the kinase activity of PI4KIIIß is necessary to prevent this unwanted lysosomal efflux under normal conditions, and to facilitate proper sorting when recycling of lysosomal material is needed, such as in the physiological context of lysosomal reformation after prolonged starvation.

Kupffer Cell Transplantation in Mice for Elucidating Monocyte/Macrophage Biology and for Potential in Cell or Gene Therapy.

Kupffer cells (KC) play major roles in immunity and tissue injury or repair. Because recapitulation of KC biology and function within liver will allow superior insights into their functional repertoire, we studied the efficacy of the cell transplantation approach for this purpose. Mouse KC were isolated from donor livers, characterized, and transplanted into syngeneic recipients. To promote cell engraftment through impairments in native KC, recipients were preconditioned with gadolinium chloride. The targeting, fate, and functionality of transplanted cells were evaluated. The findings indicated that transplanted KC engrafted and survived in recipient livers throughout the study period of 3 months. Transplanted KC expressed macrophage functions, including phagocytosis and cytokine expression, with or without genetic modifications using lentiviral vectors. This permitted studies of whether transplanted KC could affect outcomes in the context of acetaminophen hepatotoxicity or hepatic ischemia-reperfusion injury. Transplanted KC exerted beneficial effects in these injury settings. The benefits resulted from cytoprotective factors including vascular endothelial growth factor. In conclusion, transplanted adult KC were successfully targeted and engrafted in the liver with retention of innate immune and tissue repair functions over the long term. This will provide excellent opportunities to address critical aspects in the biogenesis, fate, and function of KC within their native liver microenvironment and to develop the cell and gene therapy potential of KC transplantation.

Differential gene expression in human, murine, and cell line-derived macrophages upon polarization.

The mechanisms by which macrophages control the inflammatory response, wound healing, biomaterial-interactions, and tissue regeneration appear to be related to their activation/differentiation states. Studies of macrophage behavior in vitro can be useful for elucidating their mechanisms of action, but it is not clear to what extent the source of macrophages affects their apparent behavior, potentially affecting interpretation of results. Although comparative studies of macrophage behavior with respect to cell source have been conducted, there has been no direct comparison of the three most commonly used cell sources: murine bone marrow, human monocytes from peripheral blood (PB), and the human leukemic monocytic cell line THP-1, across multiple macrophage phenotypes. In this study, we used multivariate discriminant analysis to compare the in vitro expression of genes commonly chosen to assess macrophage phenotype across all three sources of macrophages, as well as those derived from induced pluripotent stem cells (iPSCs), that were polarized towards four distinct phenotypes using the same differentiation protocols: M(LPS,IFN) (aka M1), M(IL4,IL13) (aka M2a), M(IL10) (aka M2c), and M(-) (aka M0) used as control. Several differences in gene expression trends were found among the sources of macrophages, especially between murine bone marrow-derived and human blood-derived M(LPS,IFN) and M(IL4,IL13) macrophages with respect to commonly used phenotype markers like CCR7 and genes associated with angiogenesis and tissue regeneration like FGF2 and MMP9. We found that the genes with the most similar patterns of expression among all sources were CXCL-10 and CXCL-11 for M(LPS,IFN) and CCL17 and CCL22 for M(IL4,IL13). Human PB-derived macrophages and human iPSC-derived macrophages showed similar gene expression patterns among the groups and genes studied here, suggesting that iPSC-derived monocytes have the potential to be used as a reliable cell source of human macrophages for in vitro studies. These findings could help select appropriate markers when testing macrophage behavior in vitro and highlight those markers that may confuse interpretation of results from experiments employing macrophages from different sources.