Browsing by Subject "monocyte"
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Item Open Access Antigen-Loaded Monocytes as a Novel Cancer Vaccine(2017) Huang, Min-NungDendritic cells (DC) have been the key elements in developing cancer vaccines to induce potent T cell responses to eradicate tumors. However, the common approach adopted in clinical trials using ex vivo generated DC loaded with tumor antigens (Ag) has been challenged by its limited clinical response, complexity, and quality of the manufacturing process. Alternative efforts focused on in vivo Ag loading on endogenous primary DC have not yet been well validated in their efficacy for cancer treatment, suggesting the efficiency of in vivo Ag transfer to endogenous DC from currently available Ag-delivering vehicles needs to be further improved. Here, I aim to develop an alternative cellular vaccine platform that can circumvent the aforementioned problems. I reason that classical Ly-6Chi monocytes (i.e. monocytes hereafter) can be a promising candidate to be loaded with tumor Ag and induce effective T cell responses. With advantages including easy-purification from human peripheral blood, monocytes evidently can present antigens directly via in vivo differentiation into bona fide DC or indirectly via antigen transfer to lymphoid resident DC to induce strong Th1 or cytotoxic T lymphocyte (CTL) responses. However, whether monocytes exploit favorably direct or indirect pathway to present the same Ag they are carrying to trigger effective immune responses remains unclear. Furthermore, how exactly monocytes or monocyte-derived cells transfer antigens to lymphoid resident DC has yet to be elucidated. I hypothesized that Ag-loaded monocytes can induce strong anti-tumor immunity and began the research by investigating the immune responses that can be induced by Ag-loaded monocytes. I then went on to determine the mechanisms that mediate monocyte-induced immune responses and evaluate anti-tumor efficacy of this monocyte vaccine.
In the first part of this study, I characterized the immune responses induced by Ag-loaded monocytes. By using negative selection via magnetic-activated cell sorting (MACS) columns, I was able to purify monocytes from bone marrow (BM) cells and determined that these monocytes could be successfully loaded with Ag in the forms of proteins, peptides and mRNA. I found that intravenously (IV) injected Ag-loaded monocytes induced robust Ag-specific CD4+ and CD8+ T cell responses in mice without triggering antibody responses. This vaccine activity of Ag-loaded monocytes appeared to be dose-dependent and required live monocytes with no need of ex vivo stimulation. I found that Ag-specific CD8+ T cells induced by Ag-loaded monocytes were functionally more robust than those induced by protein Ag emulsified in a traditional adjuvant CFA.
In the second part of this study, I investigated how IV injected Ag-loaded monocytes stimulate T cell responses. I identified that the spleen is the primary immune niche for Ag-loaded monocytes to induce T cell responses. I found that Ag-loaded monocytes mainly retain in the spleen where they begin to differentiate into phenotypic DC. Surprisingly, major histocompatibility complex (MHC)-deficient monocytes maintain full capacity to stimulate T cell responses, suggesting that Ag-loaded monocytes do not present Ag by themselves. I determined that endogenous splenic DC is absolutely required for monocyte-induced T cell responses. Therefore, Ag-loaded monocytes induce T cell responses indirectly via transferring Ag to splenic DC even they do differentiate into phenotypic DC in the spleen. I elucidated that this monocyte-to-DC Ag transfer occurs via gap junctions for CD8+ T cell responses and via macrophages for CD4+ T cell responses.
In the final part of this study, I demonstrated that IV injected Ag-loaded monocytes have robust anti-tumor efficacy targeting both model and validated tumor Ag in prophylactic, memory and therapeutic murine SQ melanoma models. The anti-tumor efficacy is superior to that seen with traditional adjuvants or RNA-pulsed DC vaccines, and can be combined with checkpoint blockade to increase their efficacy. Furthermore, I demonstrated that Ag-loaded monocytes have a clear anti-tumor efficacy in an intracranial glioblastoma (GBM) model targeting against mutant isocitrate dehydrogenase 1-R132H (mIDH1-R132H), a validated tumor Ag of GBM.
In conclusion, IV injection of unactivated Ag-loaded monocytes without adjuvants induces highly efficacious anti-tumor T cell responses via dual independent and efficient Ag transfer pathways to splenic DC. These findings revise the paradigm that monocytes have to be activated ex vivo to achieve optimal vaccine efficacy and reveal unappreciated cell-associated Ag acquiring pathways of splenic DCs that can be specifically manipulated for future vaccine design in the treatment of human cancers.
Item Open Access Effects of Linoleic Acid on Tether Formation between Monocytes and Endothelial Cells(2008-12-12) Irick, JoelThe fatty acid linoleic acid has been identified as a potential mediator of atherosclerotic plaque development. Treatment of monocytes with linoleic acid leads to an increase in monocyte adhesion to endothelial cells under flow conditions; however, the mechanisms through which linoleic acid affect monocyte adhesion remain unclear. Using a combination of micropipette aspiration techniques and fluorescent microscopy, I tested the hypothesis that linoleic acid increases membrane tether formation between monocytes and endothelial cells.
Treatment of U937 monocytes with free linoleic acid or albumin-bound linoleic acid reduced the cortical tension of the monocytes. The effects of albumin-bound linoleic acid on the membrane were governed by the exchange of linoleic acid from albumin to the membrane and by the removal of fatty acids from the membrane by fatty acid binding sites on albumin.
The frequency of tether formation between U937 monocytes and TNF-α stimulated HUVECs increased following treatment with free linoleic acid or albumin-bound linoleic acid. The increase in tether frequency was not due to an increase in monocyte deformability or adhesion receptor expression. Tether extraction occurred primarily through E-selectin. Treatment with free linoleic acid increased the localization of E-selectin to clathrin-coated pits suggesting an increase in the formation of nanoclusters of E-selectin on HUVECs. The increase in tether frequency was blocked by the U73122 phospholipase C inhibitor indicating that linoleic acid increased monocyte adhesion through a phospholipase C mediated mechanism.
Treatment with free linoleic acid did not affect the threshold force for tether extraction or the effective viscosity of tethers extracted from HUVECs, but it decreased the threshold force for tether extraction from U937 monocytes and increased the effective tether viscosity. Treatment with U73122 blocked the reduction in the threshold force indicating that linoleic acid affected the regulation of the membrane adhesion energy through the hydrolysis of PIP2 by phospholipase C.
The results of the study indicated that linoleic acid promoted membrane tether formation by increasing E-selectin bond formation and reducing the adhesion energy between the U937 plasma membrane and the actin cytoskeleton through the hydrolysis of PIP2 by phospholipase C.
Item Open Access Elucidating the pathogenesis of adenosine deaminase 2 deficiency: current status and unmet needs(Expert Opinion on Orphan Drugs, 2021-12-02) Tarrant, TK; Kelly, SJ; Hershfield, MSItem Open Access Elucidating the pathogenesis of adenosine deaminase 2 deficiency: current status and unmet needs(Expert opinion on orphan drugs) Tarrant, TK; Kelly, Susan J; Hershfield, Michael SIntroduction Humans have two adenosine deaminase isozymes, ADA1 and ADA2, which differ in affinity for their substrates, adenosine (Ado) and 2ʹdeoxyadenosine (dAdo), and their localization. Inherited deficiencies of ADA1 and ADA2 compromise different aspects of immune and hematological function. The metabolic consequences of ADA1 deficiency show that its enzymatic (ADA) activity is central to its biological function. By contrast, the function of ADA2 is uncertain and no direct metabolic consequences of the deficiency of ADA2 (DADA2) have yet been identified. Several potential pathogenetic mechanisms related to, or independent of, ADA2 enzymatic activity have been proposed. Areas covered We will review the discovery of ADA2 and DADA2, and two principal hypotheses: that ADA2 regulates the concentration of extracellular Ado and/or that it is a growth factor. We will consider two newer proposals that involve the effects of ADA2 products rather than its substrates, one of which postulates a pathogenic role for elevated levels of ADA2. Expert opinion Some currently proposed mechanisms of DADA2 pathogenesis are controversial or contradictory, and supportive evidence is inadequate. Progress in several areas may clarify the function of ADA2 and facilitate the development of new therapies for DADA2 based on elucidation of its pathogenesis.Item Open Access In Vitro Differentiation of Tumor- Associated Macrophages from Monocyte Precursors with Modi(2021) Kaufman, Russel; Wang, TaoTumor-associated macrophages (TAMs) are one of most important components of the tumor microenvironment. Although many assays have been developed to differentiate monocytes into macrophages (Mϕ) for studying the biology of TAMs in vitro, little is known whether the macrophages induced by these approaches can recapitulate the biology of TAMs present in the tumor microenvironment. We have developed a novel assay to differentiate human monocytes into TAMs using modified melanoma-conditioned medium, which is derived from the concentrated tumor cell culture medium. Characterization of these modified melanoma-conditioned medium-induced macrophages (MCMI-Mϕ) by multiple flow cytometry, Luminex, microarray, and immunohistochemistry analyses indicates that MCMI-Mϕ are phenotypically and functionally highly similar to the TAMs present in the tumor microenvironment.Item Open Access Mucosal Associated Invariant T (MAIT) Cell Responses Differ by Sex in COVID-19.(Med (New York, N.Y.), 2021-04-13) Yu, Chen; Littleton, Sejiro; Giroux, Nicholas S; Mathew, Rose; Ding, Shengli; Kalnitsky, Joan; Yang, Yuchen; Petzold, Elizabeth; Chung, Hong A; Rivera, Grecia O; Rotstein, Tomer; Xi, Rui; Ko, Emily R; Tsalik, Ephraim L; Sempowski, Gregory D; Denny, Thomas N; Burke, Thomas W; McClain, Micah T; Woods, Christopher W; Shen, Xiling; Saban, Daniel RSexual dimorphisms in immune responses contribute to coronavirus disease 2019 (COVID-19) outcomes, yet the mechanisms governing this disparity remain incompletely understood. We carried out sex-balanced sampling of peripheral blood mononuclear cells from confirmed COVID-19 inpatients and outpatients, uninfected close contacts, and healthy controls for 36-color flow cytometry and single cell RNA-sequencing. Our results revealed a pronounced reduction of circulating mucosal associated invariant T (MAIT) cells in infected females. Integration of published COVID-19 airway tissue datasets implicate that this reduction represented a major wave of MAIT cell extravasation during early infection in females. Moreover, female MAIT cells possessed an immunologically active gene signature, whereas male counterparts were pro-apoptotic. Collectively, our findings uncover a female-specific protective MAIT profile, potentially shedding light on reduced COVID-19 susceptibility in females.Item Open Access The role of monocyte and monocyte-derived cells in influenza-induced pathology and Th1 immune responses(2009) Lin, Kaifeng LisaMonocytes and monocyte-derived cells are important in providing innate immunity against various pathogens. Monocytes become macrophages or dendritic cells after they enter tissues during inflammation. Macrophages phagocytose microbes and kill them intracellularly in lysosomes. After macrophages are activated, they secret a variety of cytokines as part of innate defense. However, such cytokines have been implicated in causing autoimmune diseases and influenza-induced pathology. For these reasons, we have investigated the role of monocytes and monocyte-derived cells in inducing immune pathology. Moreover, monocytes are also thought to affect adaptive immunity by shaping T cell responses. Yet the enterity of their contributions to adaptive immune response remains to be determined.
CCR2 is the chemokine receptor required for inflammatory monocytes to enter tissues, and its deficiency in mice has been shown to be protective for influenza-induced immune pathology. We hypothesized that cells that depend on CCR2 to migrate into inflammaed lungs are the cells that induce immune pathology during influenza infection. First, we identified cell types that are recruited to the lungs by CCL2. Similar myeloid cell types, monocytes, monocyte-derived DCs (moDCs), and exudated macrophages (exMAC), also accumulate in the lungs during influenza infection. We then show that these myeloid cells types are derived from monocytes, and that they produce high levels of TNF-α and NOS2. Finally, we show a strong correlation between reduced accumulation of myeloid cells and decreased influenza-induced pathology and mortality in CCR2-deficient mice, suggesting that CCR2 inhibition may be a viable therapy for highly pathogenic influenza infection.
In the second part of this work, we focus on monocyte-derived dendritic cells in lymph nodes (LN). Inflammatory DCs in LN can arise from moDCs recruited via lymphatics (peripheral moDCs) and from inflammatory monocytes that enter LN directly from the blood (blood-derived moDCs). We examine the role of blood-derived moDCs in inducing LN T cell activation and polarization after immunogenic stimuli. We find that, following viral infection or immunization, inflammatory monocytes are recruited into LN directly from the blood to become CD11c+CD11bhiGr-1+ inflammatory DCs, which produce high levels of IL-12 (p70) and potently stimulate Th1 responses. This monocyte extravasation requires CCR2 but not CCL2 or CCR7. Thus, inflammatory DCs accumulation and Th1 responses are markedly reduced in CCR2-/- mice, preserved in CCL2-/- mice, and relatively increased in CCL19/21-Ser-deficient plt mice, in which all other LN DC types are reduced.
Our findings provide important insights into mutiple roles that monocytes play in both innate and adaptive immunity. Monocytes provide an early response against pathogens. As we now demonstrate, this response can be excessive, leading to a significant immune pathology during influenza infection that has been previously attributed to neutrophils. We also provide the first demonstration that monocytes play an important role in regulating adaptive immune responses. We find that monocyte-derived DCs are both sufficient and necessary for the development of Th1-polarized immune responses within LNs. Taken together, our results demonstrate that the roles played by monocytes in innate immunity adaptive immunity, and immune pathology are much greater than previously appreciated and that regulating monocyte function may be an effective means to regulate certain types of immune responses.