CD91 Derived Treg Epitope Modulates Regulatory T Lymphocyte Response, Regulates Expression of Costimulatory Molecules on Antigen-Presenting Cells, and Rescues Pregnancy in Mouse Pregnancy Loss Model.

The loss of immune tolerance to fetal antigens may result in reproductive failure. The downregulated number and activity of T regulatory lymphocytes, which are critical for the establishment of immune tolerance to fetal antigens, during pregnancy may lead to miscarriage. The adoptive transfer of Tregs prevents fetal loss in abortion-prone mice. Recently, we demonstrated that the administration of tregitopes, which are short peptides found in human and mouse immunoglobulins (IgGs), decreased the incidence of abortions in female CBA/J mice mated with DBA/2J mice. Here, two non-IgG source peptides (SGS and LKD) that can potentially bind to the major histocompatibility complex II (MHC II) with high affinity and induce Treg expansion were designed in silico. The immune dysregulation-induced pregnancy failure mouse model was used to evaluate the effect of SGS and LKD on immune response and pregnancy outcome. The fetal death rate in the SGS-treated group was lower than that in the phosphate-buffered saline-treated group. SGS and LKD upregulated the splenic pool of Tregs and modulated the T-helper cell (Th1)/Th2-related cytokine response at the preimplantation stage. Additionally, SGS and LKD downregulated the expression of CD80 and MHC class II molecules in splenic CD11c+ antigen-presenting cells. Thus, SGS treatment can result in beneficial pregnancy outcomes. Additionally, SGS peptide-mediated immunomodulation can be a potential therapeutic strategy for immune dysregulation-induced pregnancy failure.

The multifaceted role of plasminogen in inflammation.

A fine-tuned activation and deactivation of proteases and their inhibitors are involved in the execution of the inflammatory response. The zymogen/proenzyme plasminogen is converted to the serine protease plasmin, a key fibrinolytic factor by plasminogen activators including tissue-type plasminogen activator (tPA). Plasmin is part of an intricate protease network controlling proteins of initial hemostasis/coagulation, fibrinolytic and complement system. Activation of these protease cascades is required to mount a proper inflammatory response. Although best known for its ability to dissolve clots and cleave fibrin, recent studies point to the importance of fibrin-independent functions of plasmin during acute inflammation and inflammation resolution. In this review, we provide an up-to-date overview of the current knowledge of the enzymatic and cytokine-like effects of tPA and describe the role of tPA and plasminogen receptors in the regulation of the inflammatory response with emphasis on the cytokine storm syndrome such as observed during coronavirus disease 2019 or macrophage activation syndrome. We discuss tPA as a modulator of Toll like receptor signaling, plasmin as an activator of NFkB signaling, and summarize recent studies on the role of plasminogen receptors as controllers of the macrophage conversion into the M2 type and as mediators of efferocytosis during inflammation resolution.

The low density receptor-related protein 1 plays a significant role in ricin-mediated intoxication of lung cells.

Ricin, a highly lethal plant-derived toxin, is a potential biological threat agent due to its high availability, ease of production and the lack of approved medical countermeasures for post-exposure treatment. To date, no specific ricin receptors were identified. Here we show for the first time, that the low density lipoprotein receptor-related protein-1 (LRP1) is a major target molecule for binding of ricin. Pretreating HEK293 acetylcholinesterase-producer cells with either anti-LRP1 antibodies or with Receptor-Associated Protein (a natural LRP1 antagonist), or using siRNA to knock-down LRP1 expression resulted in a marked reduction in their sensitivity towards ricin. Binding assays further demonstrated that ricin bound exclusively to the cluster II binding domain of LRP1, via the ricin B subunit. Ricin binding to the cluster II binding domain of LRP1 was significantly reduced by an anti-ricin monoclonal antibody, which confers high-level protection to ricin pulmonary-exposed mice. Finally, we tested the contribution of LRP1 receptor to ricin intoxication of lung cells derived from mice. Treating these cells with anti-LRP1 antibody prior to ricin exposure, prevented their intoxication. Taken together, our findings clearly demonstrate that the LRP1 receptor plays an important role in ricin-induced pulmonary intoxications.

Immunization with the Gly1127-Cys1140 amino acid sequence of the LRP1 receptor reduces atherosclerosis in rabbits. Molecular, immunohistochemical and nuclear imaging studies.

Background: The LRP1 (CR9) domain and, in particular, the sequence Gly1127-Cys1140 (P3) plays a critical role in the binding and internalization of aggregated LDL (agLDL). We aimed to evaluate whether immunization with P3 reduces high-fat diet (HFD)-induced atherosclerosis. Methods: Female New Zealand White (NZW) rabbits were immunized with a primary injection and four reminder doses (R1-R4) of IrP (irrelevant peptide) or P3 conjugated to the carrier. IrP and P3-immunized rabbits were randomly divided into a normal diet group and a HFD-fed group. Anti-P3 antibody levels were determined by ELISA. Lipoprotein profile, circulating and tissue lipids, and vascular pro-inflammatory mediators were determined using standardized methods while atherosclerosis was determined by confocal microscopy studies and non-invasive imaging (PET/CT and Doppler ultrasonography). Studies treating human macrophages (hMΦ) and coronary vascular smooth muscle cells (hcVSMC) with rabbit serums were performed to ascertain the potential impact of anti-P3 Abs on the functionality of these crucial cells. Results: P3 immunization specifically induced the production of anti-P3 antibodies (Abs) and did not alter the lipoprotein profile. HFD strongly induced cholesteryl ester (CE) accumulation in the aorta of both the control and IrP groups, and their serum dose-dependently raised the intracellular CE of hMΦ and hcVSMC, promoting TNFR1 and phospho-NF-kB (p65) overexpression. These HFD pro-inflammatory effects were dramatically decreased in the aorta of P3-immunized rabbits and in hMΦ and hcVSMC exposed to the P3 rabbit serums. Microscopy studies revealed that P3 immunization reduced the percentage of lipids, macrophages, and SMCs in the arterial intima, as well as the atherosclerotic extent and lesion area in the aorta. PET/CT and Doppler ultrasonography studies showed that the average standardized uptake value (SUVmean) of the aorta and the arterial resistance index (ARI) of the carotids were more upregulated by HFD in the control and IrP groups than the P3 group. Conclusions: P3 immunization counteracts HFD-induced fatty streak formation in rabbits. The specific blockade of the LRP1 (CR9) domain with Anti-P3 Abs dramatically reduces HFD-induced intracellular CE loading and harmful coupling to pro-inflammatory signaling in the vasculature.

CD91 on dendritic cells governs immunosurveillance of nascent, emerging tumors.

The immune system detects aberrant, premalignant cells and eliminates them before the development of cancer. Immune cells, including T cells, have been shown to be critical components in eradicating these aberrant cells, and when absent in the host, incidence of cancer increases. Here, we show that CD91, a receptor expressed on antigen-presenting cells, is required for priming immune responses to nascent, emerging tumors. In the absence of CD91, effector immune responses are subdued, and tumor incidence and progression are amplified. We also show that, consequently, tumors that arise in the absence of CD91 express neo-epitopes with indices that are indicative of greater immunogenicity. Polymorphisms in human CD91 that are expected to affect ligand binding are shown to influence antitumor immune responses in cancer patients. This study presents a molecular mechanism for priming immune responses to nascent, emerging tumors that becomes a predictor of cancer susceptibility and progression.

Midkine drives cardiac inflammation by promoting neutrophil trafficking and NETosis in myocarditis.

Heart failure due to dilated cardiomyopathy is frequently caused by myocarditis. However, the pathogenesis of myocarditis remains incompletely understood. Here, we report the presence of neutrophil extracellular traps (NETs) in cardiac tissue of patients and mice with myocarditis. Inhibition of NET formation in experimental autoimmune myocarditis (EAM) of mice substantially reduces inflammation in the acute phase of the disease. Targeting the cytokine midkine (MK), which mediates NET formation in vitro, not only attenuates NET formation in vivo and the infiltration of polymorphonuclear neutrophils (PMNs) but also reduces fibrosis and preserves systolic function during EAM. Low-density lipoprotein receptor-related protein 1 (LRP1) acts as the functionally relevant receptor for MK-induced PMN recruitment as well as NET formation. In summary, NETosis substantially contributes to the pathogenesis of myocarditis and drives cardiac inflammation, probably via MK, which promotes PMN trafficking and NETosis. Thus, MK as well as NETs may represent novel therapeutic targets for the treatment of cardiac inflammation.

LRP1 Controls TNF Release via the TIMP-3/ADAM17 Axis in Endotoxin-Activated Macrophages.

The metalloproteinase ADAM17 plays a pivotal role in initiating inflammation by releasing TNF from its precursor. Prolonged TNF release causes many chronic inflammatory diseases, indicating that tight regulation of ADAM17 activity is essential for resolution of inflammation. In this study, we report that the endogenous ADAM17 inhibitor TIMP-3 inhibits ADAM17 activity only when it is bound to the cell surface and that cell surface levels of TIMP-3 in endotoxin-activated human macrophages are dynamically controlled by the endocytic receptor LRP1. Pharmacological blockade of LRP1 inhibited endocytic clearance of TIMP-3, leading to an increase in cell surface levels of the inhibitor that blocked TNF release. Following LPS stimulation, TIMP-3 levels on the surface of macrophages increased 4-fold within 4 h and continued to accumulate at 6 h, before a return to baseline levels at 8 h. This dynamic regulation of cell surface TIMP-3 levels was independent of changes in TIMP-3 mRNA levels, but correlated with shedding of LRP1. These results shed light on the basic mechanisms that maintain a regulated inflammatory response and ensure its timely resolution.

Overexpression of low density lipoprotein receptor-related protein 1 (LRP1) is associated with worsened prognosis and decreased cancer immunity in clear-cell renal cell carcinoma.

AIM: Clear-cell renal cell carcinoma (ccRCC) is characterized with underlying genetic disorders and the role of low density lipoprotein receptor-related protein 1 (LRP1) in ccRCC is unknown. METHOD: An in silico exploratory analysis using multiple public genetic datasets was used to establish association between LRP1 expression and clinicopathological parameters. Associations of interest were validated using 155 ccRCC samples using immunohistochemistry. RESULTS: LRP1 was overexpressed in tumor compared with normal kidney tissue. Increased LRP1 expression in ccRCC was associated with advanced stage, grade and worsened overall survival and progression-free survival. Functional annotation indicated an immune-modulatory role of LRP1 in ccRCC. LRP1 expression was significantly correlated with expressions of PBRM1, SETD2, and KDM5C. Positive correlations between LRP1 and pro-angiogenic factors ERAP1, SCG2, STAB1, and RUNX1 were observed. LRP1 expression was positively correlated with PD-L2 level. Negative correlations between LRP1 and anti-angiogenic factors EMCN and IL18 were observed. LRP1 expression was not associated with microvessel density (MVD) yet was negatively correlated with tumor-infiltrating lymphocytes (TIL). CONCLUSION: LRP1 is associated with worsened prognosis in ccRCC and is related to cancer immune modulation. LRP1-targeted therapy can be of therapeutic potential.

Low-density lipoprotein receptor-related protein 1 attenuates house dust mite-induced eosinophilic airway inflammation by suppressing dendritic cell-mediated adaptive immune responses.

BACKGROUND: Low-density lipoprotein receptor-related protein 1 (LRP-1) is a scavenger receptor that regulates adaptive immunity and inflammation. LRP-1 is not known to modulate the pathogenesis of allergic asthma. OBJECTIVE: We sought to assess whether LRP-1 expression by dendritic cells (DCs) modulates adaptive immune responses in patients with house dust mite (HDM)-induced airways disease. METHODS: LRP-1 expression on peripheral blood DCs was quantified by using flow cytometry. The role of LRP-1 in modulating HDM-induced airways disease was assessed in mice with deletion of LRP-1 in CD11c+ cells (Lrp1fl/fl; CD11c-Cre) and by adoptive transfer of HDM-pulsed CD11b+ DCs from Lrp1fl/fl; CD11c-Cre mice to wild-type (WT) mice. RESULTS: Human peripheral blood myeloid DC subsets from patients with eosinophilic asthma have lower LRP-1 expression than cells from healthy nonasthmatic subjects. Similarly, LRP-1 expression by CD11b+ lung DCs was significantly reduced in HDM-challenged WT mice. HDM-challenged Lrp1fl/fl; CD11c-Cre mice have a phenotype of increased eosinophilic airway inflammation, allergic sensitization, TH2 cytokine production, and mucous cell metaplasia. The adoptive transfer of HDM-pulsed LRP-1-deficient CD11b+ DCs into WT mice generated a similar phenotype of enhanced eosinophilic inflammation and allergic sensitization. Furthermore, CD11b+ DCs in the lungs of Lrp1fl/fl; CD11c-Cre mice have an increased ability to take up HDM antigen, whereas bone marrow-derived DCs display enhanced antigen presentation capabilities. CONCLUSION: This identifies a novel role for LRP-1 as a negative regulator of DC-mediated adaptive immune responses in the setting of HDM-induced eosinophilic airway inflammation. Furthermore, the reduced LRP-1 expression by circulating myeloid DCs in patients with eosinophilic asthma suggests a possible role for LRP-1 in modulating type 2-high asthma.

T-cell regulation through a basic suppressive mechanism targeting low-density lipoprotein receptor-related protein 1.

Cell adhesion is generally considered to depend on positive regulation through ligation of integrins and cytokine receptors. However, here we show that T-cell adhesion, and notably also T-cell receptor (TCR) -induced activation, are subject to constant suppression through shedding of low-density lipoprotein receptor-related protein 1 (LRP1). The broad-spectrum metalloprotease inhibitor GM6001 abrogated shedding, so inducing prominent cell surface expression of LRP1 while enhancing TCR-induced activation and adhesion to ß1 and ß2 integrin ligands, hence arresting the cells. Integrin ligands also inhibited shedding but the effect was less potent than that of GM6001. Unlike GM6001, integrin ligands also induced cell surface expression of full-length thrombospondin-1 (TSP170) and TSP130, which associated with LRP1, and TSP110, which did not associate with LRP1. Cell surface expression of LRP1 and TSP130 were induced exclusively in adhering cells, expression of TSP110 preferentially in non-adhering cells and expression of TSP170 correlated with T-cell motility. The pro-adhesive chemokine CXCL12 also inhibited LRP1 shedding and induced surface expression of TSP170 and TSP130 while inhibiting TSP110. Exogenous TSP-1 and ligation of CD28 inhibited shedding although less effectively than GM6001, and the inhibition through CD28 was independent of TSP-1. Small interfering RNA silencing experiments confirmed involvement of LRP1 and TSP-1 in integrin-dependent adhesion and TCR-induced activation. Hence, the poor LRP1 expression in T cells depends on shedding. Integrin ligands and CXCL12 antagonize shedding through a TSP-1-dependent pathway and ligation of CD28 antagonizes shedding independent of TSP-1. The disappearance of LRP1 from the cell surface may provide basic immunosuppression at the T-cell level.

Phenotypically distinct helper NK cells are required for gp96-mediated anti-tumor immunity.

A number of Heat Shock Proteins (HSPs), in the extracellular environment, are immunogenic. Following cross-presentation of HSP-chaperoned peptides by CD91(+) antigen presenting cells (APCs), T cells are primed with specificity for the derivative antigen-bearing cell. Accordingly, tumor-derived HSPs are in clinical trials for cancer immunotherapy. We investigate the role of NK cells in gp96-mediated anti-tumor immune responses given their propensity to lyse tumor cells. We show that gp96-mediated rejection of tumors requires a unique and necessary helper role in NK cells. This helper role occurs during the effector phase of the anti-tumor immune response and is required for T cell and APC function. Gp96 activates NK cells indirectly via APCs to a phenotype distinct from NK cells activated by other mechanisms such as IL-2. While NK cells have both lytic and cytokine producing properties, we show that gp96 selectively activates cytokine production in NK cells, which is important in the HSP anti-tumor immune response, and leaves their cytotoxic capacity unchanged.

Potential role of heat-shock protein 70 and interleukin-15 in the pathogenesis of threatened spontaneous abortions.

PROBLEM: The role of HSP70 and both its constitutive (Hsc) and inducible (Hsp) forms in the pathogenesis of threatened spontaneous abortions was investigated. METHOD OF STUDY: Immunohistology and/or immunofluorescence was used to analyze paraffin-embedded tissue sections, and reverse transcriptase-quantitative polymerase chain reaction and flow cytometry were used for analyses of decidual mononuclear cells (DMCs) and confocal microscopy for the detection of perforin, granulysin, and lysosome-associated membrane protein-1 (LAMP-1) in decidual lymphocytes (DLs). RESULTS: The percentage of single Hsp70(+) , Hsc70(+) , and IL-15(+) cells and mRNA levels of HSP70, CD91, and TLR4 were lower in the decidua basalis in cases of threatened miscarriages compared to that in cases of normal pregnancy. In a suspension of normal DMCs, IL-15 significantly decreased the HSP70 members and TLR4 in dendritic cells, T cells, and NK cells while increasing CD91 in NK cells alone. CONCLUSION: Downregulation of Hsc70, Hsp70, and IL-15 expression at gene and/or protein levels might support the retention of fertilization products in cases of missed abortion and blighted ovum.

Low-density lipoprotein receptor-related protein 1 mediates α1-antitrypsin internalization in CD4+ T lymphocytes.

In α1-antitrypsin-deficient HIV patients, an accelerated decline of CD4(+) T cell numbers is observed, suggesting that α1-antitrypsin is a potential endogenous HIV inhibitor. In infected T lymphocytes, α1-antitrypsin potently blocks NF-κB activation and HIV-1 replication by directly interacting with IκBα in the cytosol, thereby altering its ubiquitination pattern. However, the mechanism of α1-antitrypsin entry into the cytosol, where IκBα locates, remains unclear. In the present study, we investigated the mechanism of α1-antitrypsin internalization in CD4(+) T cells. Thus, primary CD4(+) T cells were infected with HIV-1 and then incubated with α1-antitrypsin to detect its internalization. We found that CD4(+) T cells internalized α1-antitrypsin through a clathrin-dependent endocytosis process. Next, intracellular α1-antitrypsin exerted the inhibitory effect on NF-κB activation and HIV-1 replication. On primary CD4(+) T cells, α1-antitrypsin interacted with low-density lipoprotein receptor-related protein 1 to initiate the internalization. Inside CD4(+) T lymphocytes, α1-antitrypsin was transported from the endosome to the lysosome and then released into the cytosol, where it is possible for α1-antitrypsin to directly interact with IκBα. These results together suggest that α1-antitrypsin internalization is a clathrin-dependent and low-density lipoprotein receptor-related protein 1-mediated endocytosis process. Internalized α1-antitrypsin is transported through the endosome-lysosome-cytosol routine to interact with cytosolic IκBα and block NF-κB activation and HIV-1 replication.

Heat-Shock Proteins 70 Induce Pro-Inflammatory Maturation Program in Decidual CD1a(+) Dendritic Cells.

PROBLEM: The aim of the study was to assess possible binding of a mixture of constitutive Hsc70 and inducible Hsp70 forms (HSP70) to Toll-like receptor (TLR) 4 and CD91 receptors on decidual CD1a(+) dendritic cells (DCs) and their influence on DCs maturation status. METHOD OF STUDY: Immunohistology and immunofluorescence of paraffin-embedded first trimetester and term pregnancy decidua were performed together with flow cytometry detection of antigens in DCs after stimulation of decidual mononuclear cells with HSP70. RESULTS: Hsc70 and Hsp70 labeling revealed intracellular and nuclear staining in trophoblast cells. The numbers of Hsc70(+) and Hsp70(+) cells of decidual tissue were higher in early pregnancy decidua than in decidua at term. HSP70 binds CD91 and TLR4 receptors on CD1a(+) DCs and increased the expression of CD83, HLA-DR, CD80, and CD86, but decreased CC receptor (CCR) 5. HSP70 increased CC ligand (CCL) 3 and CCL22. HSP70 in the concentration of 1 µg/mL increased the percentage of interferon-γ and interleukin (IL)-15-expressing cells over the cells expressing IL-4. CONCLUSION: HSP70 binds CD91 and TLR4 on decidual CD1a(+) DCs, causes their maturation, and increases IL-15 in the context of Th1 cytokine/chemokine domination, which could support immune response harmful for ongoing pregnancy.

Bright expression of CD91 identifies highly activated human dendritic cells that can be expanded by defensins.

CD91 is a scavenger receptor expressed by different immune cells and its ligands defensins have been demonstrated to contribute to immune responses against infections and tumours. We previously demonstrated that CD91 is expressed on human monocyte-derived dendritic cells (moDCs) and that human defensins stimulate in vitro the activation of these cells. In this study, we observed that CD91 is expressed at different levels on two distinct moDC subsets: CD91(dim) and CD91(bright) moDCs. Although CD91(bright) moDCs represented a small proportion of total moDCs, this subset showed higher levels of activation and maturation markers compared with CD91(dim) moDCs. The frequency of CD91(bright) moDCs increased by ~ 50% after in vitro stimulation with recombinant human neutrophil peptide-1 (rHNP-1) and recombinant human ß defensin-1 (rHBD-1), while lipopolysaccharide (LPS) stimulation decreased it by ~ 35%. Both defensins up-regulated moDC expression of CD80, CD40, CD83 and HLA-DR, although to a lower extent compared with LPS. Notably, upon culture with rHNP-1 and rHBD-1, CD91(bright) moDCs maintained their higher activation/maturation status, whereas this was lost upon culture with LPS. Our findings suggest that defensins promote the differentiation into activated CD91(bright) DCs and may encourage the exploitation of the CD91/defensins axis as a novel therapeutic strategy to potentiate antimicrobial and anti-tumour immune response.

Antigen-induced regulation of T-cell motility, interaction with antigen-presenting cells and activation through endogenous thrombospondin-1 and its receptors.

Antigen recognition reduces T-cell motility, and induces prolonged contact with antigen-presenting cells and activation through mechanisms that remain unclear. Here we show that the T-cell receptor (TCR) and CD28 regulate T-cell motility, contact with antigen-presenting cells and activation through endogenous thrombospondin-1 (TSP-1) and its receptors low-density lipoprotein receptor-related protein 1 (LRP1), calreticulin and CD47. Antigen stimulation induced a prominent up-regulation of TSP-1 expression, and transiently increased and subsequently decreased LRP1 expression whereas calreticulin was unaffected. This antigen-induced TSP-1/LRP1 response down-regulated a motogenic mechanism directed by LRP1-mediated processing of TSP-1 in cis within the same plasma membrane while promoting contact with antigen-presenting cells and activation through cis interaction of the C-terminal domain of TSP-1 with CD47 in response to N-terminal TSP-1 triggering by calreticulin. The antigen-induced TSP-1/LRP1 response maintained a reduced but significant motility level in activated cells. Blocking CD28 co-stimulation abrogated LRP1 and TSP-1 expression and motility. TCR/CD3 ligation alone enhanced TSP-1 expression whereas CD28 ligation alone enhanced LRP1 expression. Silencing of TSP-1 inhibited T-cell conjugation to antigen-presenting cells and T helper type 1 (Th1) and Th2 cytokine responses. The Th1 response enhanced motility and increased TSP-1 expression through interleukin-2, whereas the Th2 response weakened motility and reduced LRP1 expression through interleukin-4. Ligation of the TCR and CD28 therefore elicits a TSP-1/LRP1 response that stimulates prolonged contact with antigen-presenting cells and, although down-regulating motility, maintains a significant motility level to allow serial contacts and activation. Th1 and Th2 cytokine responses differentially regulate T-cell expression of TSP-1 and LRP1 and motility.

Antibody-coupled monolithic silica microtips for highthroughput molecular profiling of circulating exosomes.

Exosome-mediated signal transportation plays a variety of critical roles in cancer progression and metastasis. From the aspect of cancer diagnosis, circulating exosomes are ideal resources of biomarkers because molecular features of tumor cells are transcribed on them. However, isolating pure exosomes from body fluids is time-consuming and still major challenge to be addressed for comprehensive profiling of exosomal proteins and miRNAs. Here we constructed anti-CD9 antibody-coupled highly porous monolithic silica microtips which allowed automated rapid and reproducible exosome extraction from multiple clinical samples. We applied these tips to explore lung cancer biomarker proteins on exosomes by analyzing 46 serum samples. The mass spectrometric quantification of 1,369 exosomal proteins identified CD91 as a lung adenocarcinoma specific antigen on exosomes, which was further validated with CD9-CD91 exosome sandwich ELISA measuring 212 samples. Our simple device can promote not only biomarker discovery studies but also wide range of omics researches about exosomes.

Properties of human blood monocytes. I. CD91 expression and log orthogonal light scatter provide a robust method to identify monocytes that is more accurate than CD14 expression.

BACKGROUND: This study was designed to improve identification of human blood monocytes by using antibodies to molecules that occur consistently on all stages of monocyte development and differentiation. METHODS: We examined blood samples from 200 healthy adults without clinically diagnosed immunological abnormalities by flow cytometry (FCM) with multiple combinations of antibodies and with a hematology analyzer (Beckman LH750). RESULTS: CD91 (α2 -macroglobulin receptor) was expressed only by monocytes and to a consistent level among subjects [mean median fluorescence intensity (MFI) = 16.2 ± 3.2]. Notably, only 85.7 ± 5.82% of the CD91(+) monocytes expressed high levels of the classical monocyte marker CD14, with some CD91(+) CD16(+) cells having negligible CD14, indicating that substantial FCM under-counts will occur when monocytes are identified by high CD14. CD33 (receptor for sialyl conjugates) was co-expressed with CD91 on monocytes but CD33 expression varied by nearly ten-fold among subjects (mean MFI = 17.4 ± 7.7). In comparison to FCM analyses, the hematology analyzer systematically over-counted monocytes and eosinophils while lymphocyte and neutrophil differential values generally agreed with FCM methods. CONCLUSIONS: CD91 is a better marker to identify monocytes than CD14 or CD33. Furthermore, FCM (with anti-CD91) identifies monocytes better than a currently used clinical CBC instrument. Use of anti-CD91 together with anti-CD14 and anti-CD16 supports the identification of the diagnostically significant monocyte populations with variable expression of CD14 and CD16.