In vitro differentiation of human myeloid cell populations.

Abstract (Swedish)

The human immune system responds to a wide variety of pathogens and microorganisms to defend the body. Investigating the cell types and molecular markers of the immune system are critical for the understanding of diseases and finding treatments. Antigen presenting cells (APCs) are sentinels of the immune system that sample the surrounding environment and educate the components of the adaptive immune system such as T-cells and B-cells. The low frequency of APC in blood limits the possibilities to perform research studies that require large number of cells. In vitro models that mimic the in vivo counterparts are valuable alternatives, as they can be differentiated from progenitor cells in higher numbers, and has an extended lifetime compared to differentiated cells received from blood. In this study, we focus on the differentiation of monocytes into dendritic cells (DCs) and macrophages by cultivating the cells with two different setups of cytokines containing M-CSF/IL-4/TNF-alpha and GM-CSF/IL-4. DCs and macrophages express different receptors and have different functions in the body depending on their location, cell subset, disease context and immune microenvironment. We have prepared two panels for flow cytometry consisting of DC, macrophage and monocyte associated molecular markers to be able to distinguish these cell populations amongst our differentiated and activated cells. These markers were investigated on our differentiated cells to distinguish changes in expression as a result of our in vitro culturing with the two setups of cytokines. In this study we compare and analyze these two differentiation protocols according to the molecular markers on cells at different times during differentiation. Result showed GM-CSF/IL-4 protocol had better effect on moDC induction, and the expression of CCR7, CD163, TRAIL and CD88 were significantly increased after differentiation on moDC in both protocols, while moDC expressed more CD1c after differentiation in both protocols. These acquired moDCs are marked by high expression of CD1a, CD1c, CD11b, CD11c and HLA-DR, expression of CD88 and extremely low expression of CCR7 and CCR2, no expression of CD14, CD163 and CD16, whose phenotype is proved similar to that of inflammatory DC.

Popular Abstract

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The immune system is a defense network for our body to respond against outside invaders including bacteria, fungi and viruses, and protect us from diseases by, for instance, removing infected or abnormal cells. The innate immune system acts as a rapid response system for recognizing foreign, potentially dangerous agents, and activating the adaptive immune system to produce molecules specifically towards certain proteins, so-called, antigen. Antigen-presenting cells (APCs), including macrophages and dendritic cells, can act as sentinels of the immune system and thus play an important role in collecting, processing and presenting specific fragments of pathogens and initiating adaptive T and B cell responses, thus connecting the innate and adaptive immune systems. When the adaptive immune system is activated, it is far more effective in defending the body against infection than the innate immune system. DCs are therefore of great interest for understanding the intricacies of immunity and disease.

In some cases, the immune system is not adequately activated, and the functionality of DCs is not sufficient to mount a proper immune response against disease, which can lead to progression of diseases. APCs is a very heterogenous group of immune cells, and recent technological breakthroughs, such as single-cell sequencing, have shed light on the complexity within this population of cells. They are currently intensively studied in the context of various diseases, such as cancer, to find means to increase their activity for enhanced T cell activation. APCs are very scarce in the body, making studies on human primary APCs difficult. As a consequence, APCs generated outside human body are a valuable alternative, and these cellular models are frequently used to investigate the functions and phenotypes of different macrophages and DCs during different conditions and disease settings. For instance, this in-vitro models can be used to assess their differentiation, responses to cytokines and functional properties, and they are also used as cellular sources in various therapeutic cancer vaccines.

In our project, we conducted experiments to produce DCs and macrophages from another immune cell type called peripheral blood monocytes. Monocytes exist in large amounts in blood and are therefore easy to isolate and culture. Monocytes were isolated using magnetic particles coated with specific antibodies targeted to attach to monocytes. Subsequently, production of DCs was performed by culturing monocytes together with a cocktail of cell-to-cell communication molecules called cytokines, resulting in monocytes differentiating into DCs, termed monocyte-derived DCs (moDCs) and macrophages, termed monocyte-derived macrophages (moMACs). The aim of our project was to compare the effect of different mixes of cytokines on the differentiation of monocytes into macrophages and DCs. Multiparameter flow cytometry was used to assess the cellular changes after stimulation with 2 different cytokine cocktails.

The results showed that one of our cytokine cocktails was more effective at stimulating DC differentiation than the other. These cocktails contained cytokines called IL-4 and GM-CSF. Acquired moDCs had the high expression of cell surface molecules such as, CD1a, CD1c, CD11b, CD11c and HLA-DR, and they did not express CD14, CD163 and CD16, which are considered to resemble inflammatory DCs.

• author: Li, Ziyi and Gummeson, Noomi
• supervisor: Can Altunbulakli; Malin Lindstedt
• organization: Department of Immunotechnology; Educational programmes, LTH
• course: KIMM01 20221
• year: 2022
• type: H2 - Master's Degree (Two Years)
• subject: Biology and Life Sciences
• keywords: monocyte-derived DCs, monocyte-derived macrophages, immunotechnology
• language: English
• id: 9100796
• date added to LUP: 2022-09-26 09:14:50
• date last changed: 2022-09-26 09:14:50