Internalisation of antigen-adjuvant conjugate in human dendritic cells: An assay development for using live cell imaging

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Internalisation of antigen-adjuvant conjugate in human dendritic cells

An assay development for using live cell imaging

Linnéa Gustafsson

Degree Project in Biologicals and Immunotherapy, 30 credits, autumn semester 20

Supervisor: Ida Olsson Examiner: Greta Hultqvist

Division for Immune oncology

Department of Pharmaceutical Biosciences Faculty of Pharmacy

Uppsala University

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Populärvetenskaplig sammanfattning

Cancerterapier har under många år endast bestått av kirurgi, strålning och cellgifter. Under de senaste årtiondena har en ny form av cancerterapi utvecklats, immunoterapi. Immunoterapier går ut på att få kroppens egna immunförsvar att effektivt angripa cancern. Den här typen av terapier är mer specifikt mot tumörcellerna och leder då till färre biverkningar än de

traditionella terapiformerna. Cancervaccin är en del av immunoterapin idag. Till skillnad från vaccin mot virus förebygger inte cancervacciner uppkomsten av cancer utan det är en

behandlingsmetod. Cancervaccin består av en del av tumören, en peptid, och ett adjuvans som förbättrar effekten av peptiden. För att peptiden ska kunna verka måste det bearbetas och brytas ner i mindre delar, detta sker inuti dendritceller (DC) som är en del av immunförsvaret.

DC kan i sig inte attackera tumörceller utan presenterar delar av peptiden till T-celler som i sin tur attackerar tumörcellerna.

Den här studien gick ut på att utveckla en metod för att titta på upptaget av en peptid och adjuvans i DC, när frisläppning av peptiden sker och effekten av adjuvantet. Ett mikroskop användes för att titta på DC i realtid och samtidigt ta bilder. För att kunna följa DC under en längre tid behövs en beläggning som gör att de ligger still och inte åker utanför bildfokus. I studien prövades tre beläggningar, kollagen typ I, fibronektin och matrigel, i olika

koncentrationer. Beläggningen får inte aktivera DC då det påverkar upptaget av peptiden.

Resultatet i studien visade att matrigel i hög koncentration var den bäst lämpade beläggningen då det immobiliserade cellerna utan att aktiverade dem.

Innan den utvecklade metoden kan användas med cancervaccinet behöver optimering ske i mikroskoperings metoden. Cellerna befinner sig i vätska under mikroskoperingen, den här vätskan avdunstade med tiden. Om cellerna befinner sig i för lite vätska kan det påverka studiens resultat eftersom cellernas påverkas negativt.

Genom att studera upptaget av cancervaccinet och frisläppningen av peptiden kan cancervaccinet utvecklas ytterligare utifrån resultatet. Ju mer processerna bakom

cancervaccinet studeras desto mer information finns om cancervaccinet. Ju mer information som finns om ett cancervaccin desto bättre går det att optimera det ur både effekt och säkerhetssynpunkt.

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Abstract

Introduction: Cancer vaccines are a therapeutic approach to initiate an antigen specific cytotoxic immune responses against tumors. Cancer vaccines are composed by an antigen (tumor peptide) and adjuvant. A peptide in combination with adjuvants effectively activate dendritic cells (DCs), the most efficient antigen presenting cells in our immune system. DCs prime and activate CD8+ cytotoxic T cells which generates an antigen specific response.

Aim: Developing an assay to study the internalisation rout of an antigen-adjuvant conjugate in human dendritic cells by using live cell imaging.

Method: Immobilisation of cells is necessary for the ability to perform live cell imaging for several hours. The immobilisation ability of three coatings, collagen type I, fibronectin and matrigel, at different concentrations were evaluated by using live cell imaging in a

fluorescence microscope. The potential induction of activation of the cells were evaluated by using flow cytometry and ELISA.

Results: Immature DCs internalise antigen-adjuvant conjugate more efficiently than mature and activated DCs. Therefore, it is important that the coating do not induce activation. Cells must also be immobilised for the possibility of long term detection. Collagen type I

immobilised cells and induced activation in all investigated concentrations. Fibronectin and matrigel had concentration-dependent abilities to immobilise the cells. Matrigel did not activate the cells whilst fibronectin was concentration dependent.

Conclusion: Matrigel immobilise the cells which enables long term single cell imaging without activation.

Keywords: cancer vaccine, coating, dendritic cells, immobilisation, internalisation, live cell imaging

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Abbreviations

APC, antigen presenting cell DC, dendritic cell

ER, endoplasmic reticulum

FACS, Fluorescence-Activated Cell Sorting LPS, lipopolysaccharides

mAbs, monoclonal antibodies

MHC, major histocompatibility complex MoDC, monocyte-derived dendritic cell PBMC, peripheral blood mononuclear cell TC, tissue culture

TCR, t cell receptor

TNFR, tumor necrosis factor receptor

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1. Introduction

1.1 Dendritic cells

Dendritic cells (DC) are the most efficient antigen presenting cells (APC) in our immune system. Antigens are components on the bacterial wall, such as lipopolysaccharides (LPS), viral products, double-stranded RNA, or inflammatory stimuli such as cytokines. To be able to present extracellular antigens to other immune cells, DCs must internalise the antigens.

Receptor mediated endocytosis is one mechanism of internalisation (1,2). CD40, a tumor necrosis factor receptor (TNFR) present on all APCs, is an example of a surface receptor that mediates endocytosis (3). Ligation of costimulatory molecules, such as CD40, is one mechanism that induces maturation of DCs (4–6). DC maturation can also be triggered by encountering antigens. However, antigens do not always induce complete maturation of DCs which results in anergic T cells (5,7). A complete maturation of DC is therefore necessary for a successful activation of T cells. A mature DC upregulates Major Histocompatibility Complex (MHC) molecules and co stimulatory molecules on the cell surface and secrete different cytokines, such as IL-12. The maturation enables a specific antigen presentation to both CD4+

helper T cells and CD8+ cytotoxic T cells (2,8).

1.1.1 MHC class I and II

The origin of the antigens plays a role in the type of MHC class the antigen will be presented on. Extracellular antigens are internalised, digested into peptides and loaded on MHC class II in endocytic compartments. The MHC-class-II-peptide complex is then transported to the cell surface where the peptide is presented to CD4+ helper T cells (2).

MHC class I present peptides from intracellular proteins, self or non self. Non self-antigens can be generated from a viral infection. Self-antigens can be produced by tumor cells and can trigger antitumor responses (1). These intracellular antigens are degraded into peptides in the cytosol by the proteasome. The peptides are then transported to the endoplasmic reticulum (ER) where the peptide is loaded onto newly produced MHC class I molecules. The MHC-class-I-peptide complex are then exported to the cell surface through the Golgi apparatus. MHC class I presents peptides to CD8+ cytotoxic T cells (1,8).

1.1.2 Cross presentation

All nucleated cells express MHC class I molecules, antigens can be presented on all cells.

However, not all cells are capable of activating resting naïve T cells and therefore not capable

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of initiating a cytotoxic immune response. Through a process called cross presentation, extracellular antigens can be presented on MHC class I molecules on APCs to provide a cytotoxic immune response (1,2). The CD40 pathway enhances the cross-presentational rout when targeted together with an antigen (3,9). There are two main intracellular pathways for cross presentation, the cytosolic- and the vacuolar pathway (1,2). In the cytosolic pathway, antigens are internalised by DCs, similar to MHC class II antigen presentation but the antigens are transported out of the phagosome and into the cytosol where the antigen is degenerated by the proteasome. The peptide can then either be loaded onto MHC class I the classical way, described above or be transported into the phagosome again and loaded on MHC class I there.

In the vacuolar pathway, the antigen is degenerated and loaded onto the MHC class I molecule in the phagosome (1,8).

1.1.3 T cell activation signals

There are three essential signals APC have to provide to the T-cells for antigen specific T cell activation (10). Signal one is the presentation of peptides on MHC molecules on DCs to T cell receptors (TCR) on T cells. This signal provides T cell recognition of the specific antigen.

Signal two is the interaction of costimulatory molecules on the cell surface, on both APC and T cells. This interaction activates the T cell and induces an expansion in number of the T cell population. The last and third signal is cytokine release by APC. Cytokines directs the differentiation of T cells into different effector T cell subtypes depending on what cytokines are secreted from APC (10–12).

1.2 Cancer vaccine

There are significant differences between cancer vaccines and conventional vaccines for infectious diseases. Due to the heterogeneity in tumor types and progression it is generally not possible to use prophylactic vaccines against cancer tumors. Cancer vaccines are therefore a therapeutic approach. A cancer patients’ immune system is dysfunctional due to both tumor- specific mechanisms and conventional cancer therapies, such as radiation and chemotherapies.

In the process of eliminating rapidly dividing neoplastic cells, the conventional cancer therapies also target self-replicating immune cells. Tumors contribute to the dysfunctional immune system by suppressive mechanisms and by redirecting immune cells (11). Cancer vaccines are a way of circumvent these suppressive effects generated from the tumor. These vaccines are therefore a promising alternative or combination to conventional cancer treatment such as radiation and chemotherapies (11,13)

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1.2.1 Adjuvants

Cancer peptide vaccines alone have a low efficacy (2.6 %) and therefore an adjuvant is desirable (14). As mentioned before, DC maturation causes upregulation of costimulatory molecules and cytokine secretion, two of the three essential signals to activate T cells (7,10). However, antigen presentation by incompletely activated DCs, that does not provide either signal two or three, causes anergic T cells (7). Adjuvants are additional components to a vaccine that in combination with antigen enhances the immunogenicity by contributing to fully activated DCs that provide all three essential signals to the T cells (5,15). Adjuvants can also function as antigen transporters to enhance uptake of the APC and improve antigen specific response (13). Immune stimulatory monoclonal antibodies (mAbs) as adjuvants improves the performance of APCs and boost anticancer responses. Antigen carrying anti CD40 mAbs have been shown to effectively activate DCs to prime T-cells, and provide all three essential signals (4,16). The autoimmune events that are commonly observed with checkpoint antibodies, CTLA-2 and PD- 1/PD-L1 mAbs, are not or rarely observed with CD40 mAbs (17).

1.3 Aim

The aim of this project was to develop an assay to study internalisation and recycling pattern of an antigen-adjuvant conjugate in human dendritic cells by using live cell imaging. The three main questions the assay should be able to answer are:

• At what time point is the adjuvant internalised?

• Where and when is the peptide released?

• Is the antibody recycled after peptide release?

2. Method

2.1 Cell culturing

2.1.1 Culturing of THP-1 cells

THP-1 cells were cultured in THP-1 cell culture media (RPMI Medium 1640 (1X) + GlutaMAX™-l (gibco), 10% FBS (Gibco) and 1% PeSt (Life Technologies) in a 6 well Tissue culture (TC) plate (Starstedt) and in a TC Flask (T75 vent. Cap, Starstedt). The cells were cultured in a density of 2-8 x 10⁴cells/ml and the media were changed every 2-3 days.

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2.1.2 Isolation of monocytes

Buffy coats from healthy blood donors were purchased from the Academic hospital in Uppsala. Peripheral blood mononuclear cells (PBMCs) were isolated by diluting the buffy coats with PBS (Dulbecco’s Phosohate Buffered Saline, Biowest) and using Ficoll-Plaque medium (Cytiva) as a density gradient in a SepMate™ tube (STEMCELL Technologies).

When the SepMate™ tube, containing all components, was centrifuged at 1200 xg for 10 minutes, plasma and PBMC were separated from erythrocytes and granulocytes by the density gradient medium. The plasma and PBMC was transferred to a falcon tube and the PBMCs were washed with PBS. The PBMCs were mixed with CD14 microbeads (Miltenyi Biotec) and incubated for 15 minutes in 4°C. The PBMC and microbead suspension were run through a LC colon (Miltenyi Biotec) attached to a MACS multistand magnet (SN: 48147, Miltenyi Biotec). The CD14+ cells were collected in a falcon tube.

2.1.3 Differentiation of monocytes to MoDCs

To produce immature monocyte-derived dendritic cells (MoDCs), the CD14+ cells were diluted in complete culture medium (RPMI Medium 1640 (1X) + GlutaMAX™-l, 10% FBS, 1% PeSt and 1% Hepes (Gibco)) to a concentration of 1x10⁶ cells/ml. A volume of 10 ml cell- suspension were cultured in a TC-dish (100 x 20 mm) (Starstedt) at 37°C in 5% CO2 for six days. The CD14+ cells were stimulated with 75ng/ml hGM-CSF (PeproTech) and 50 ng/ml hIL-4 (PeproTech) on day 0, 3 and 5. The CD14+ cells were analysed with flow cytometry using a Cytoflex (Beckman Coulter) before and after stimulation to investigate the quality of the differentiation.

2.2 Coating procedure

The following concentrations of collagen type I, fibronecin and matrigel were used in the different assays.

Table 1. Coating concentrations used for the immobilisation assay

Collagen type I (μg/cm²)

Fibronectin (μg/cm²)

Matrigel (mg/ml)

10 10 4

5 5 1

1 1 0.75

0.5 0.5 0.5

0.1

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Table 2. Coating concentrations used for the activation assay.

Collagen type I (μg/cm²)

Fibronectin (μg/cm²)

Matrigel (mg/ml)

10 10 8

5 5 6

1 1 4

0.5 0.5

2.2.1 Collagen type 1

Collagen type 1 was diluted in sterile ddH2O to four concentrations between 0.5-10 µg/cm² (18). A volume of 100 µl diluted collagen were added to the wells of a 96 well TC plate (Starstedt) and were incubated at 4°C over night. After incubation the collagen solutions were discarded and the wells were washed with 150 µl PBS before cells were plated.

2.2.2 Matrigel

Matrigel was thawed on ice at 4°C over night. Matrigel were diluted in cold complete MoDC cell culture media (composition previously described) to seven different concentrations between 0.1-8 mg/ml. A volume of 35 µl of diluted matrigel were added to the 96 well TC plate (Starstedt) and incubated in 37°C for two hours. The matrigel were washed with 100 µl PBS. If the matrigel was not used directly, 100 µl of PBS was added and stored in 4°C over night. The matrigel were dissolved in 28 µl dispase (Corning) before analysis with Cytoflex.

2.2.3 Fibronectin

Fibronectin was thawed on ice at 4°C over night. Fibronectin were diluted in cold PBS to four different concentrations between 0.5-10 10 µg/cm² (18). A volume of 100 µl diluted

fibronectin were added to the wells of a 96 well TC plate (Starstedt) and were incubated at 4°C over night. The fibronectin solutions were discarded and the wells were washed with 150 µl PBS before cells were plated.

2.3 Analysis of immobilisation and activation

2.3.1 Live cell imaging

For live cell imaging an ImageXpress Micro XLS (Molecular devices) microscope were used.

MoDCs in complete cell media were plated on a coated 96 well assay plate with clear bottom (Corning Incorporated, Costar) for analysis in ImageXpress.

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2.3.1.1 Staining

MoDCs were stained with Hoechst 33342 (Thermo scientific) before imaging. The Hoechst stain were diluted in PBS to 1 µg/ml and 50 µl of the solution were added to each well in the 96 well plate containing cells that should be analysed with imageXpress. The cells were stained in room temperature for 20 min in the dark. After incubation the plate was centrifuged and the Hoechst solution was discarded and the cells were diluted in complete cell media.

The antibody HLA-A2-FITC (BB7.2, BioLegend) were used for the membrane staining of THP-1 cells. The wells were stained for 20 minutes in room temperature and in the dark. The cells were then washed with PBS and diluted in THP-1 cell media.

2.3.2 Flow cytometry assay

The MoDCs were stained with the following antibodies from BioLegend, HLA-DR-PE-Cy5 (I.243), CD86-BV421 (IT2.2), CD83-APC (HB15e), CD1a-PE (HI149), CD14-APC-Cy7 (63D3) and CD40-FITC (5C3). The cells were stained for 20 minutes in 4° C in the dark. The wells were washed with PBS (1% BSA) and then re-suspended in 100 µl Fluorescence- Activated Cell Sorting (FACS) buffer (PBS and 3mM EDTA (VWR)). The flow cytometry analysis was performed with Cytoflex and the raw data were processed with Kaluza Analysis Software version 2.1 and GraphPad Prism software version 7.04.

2.3.3 IL-12 p40 ELISA assay

A high binding ELISA plate (Sarstedt) and a ELISA MAX™ kit (Lot: B268986, Biolegend) were used to perform an ELISA assay. The assay were preformed according to

manufacturer’s instructions (19). In short: All incubations occurred in room temperature on a shaking table (500 rpm) and were followed by washing in PBS (0.05% Tween-20 (Sigma)).

The ELISA plate were coated with a capture antibody overnight and then blocked for one hour. The samples and standard were loaded on the plate and incubated for two hours. A detection antibody was added and incubated for one hour whereupon Avidin-HRP were incubated for 30 minutes. Lastly, TMB substrate was added and incubated in dark until IL-12 positive wells turned intensely blue. The reaction was stopped with 1M H2SO4 and the absorbance were measured at 450 nm with a microplate reader (Fluostar Omega 415-1734 (BMGlabtech)). The raw data from the microplate reader were further processed with GraphPad Prism and the statistical analysis was performed in the same software.

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3. Results

3.1 Cell density optimisation

DCs accounts for 1-2% of all PBMCs in a blood sample. It is possible to use MoDCs when preforming in vitro studies since MoDCs have DC-characteristics. MoDCs are derived from CD14+ monocytes which accounts for 5-10% of the PBMCs in a blood sample (20,21). THP- 1 cell line is a human monocytic leukaemia cell line which is more stable and easier to culture than MoDCs (22). THP-1 cells are used for method development before experiments preformed on MoDCs due to the six-day differentiation of CD14+ monocytes to MoDCs.

To be able to follow the path of the antigen-adjuvant conjugate in single cells the density of the cells must not be too dense. At a high density multiple cells might overlap. Out of the five densities images for THP-1 cells, the two highest densities were the most promising (figure 1). The figure shows that there are more cells overlapping at 5x10⁴ cells/well than at 2.5x10⁴ cells/well. However, one has to take into account for cell loss at staining with steps such as centrifugation and supernatant discarding which is why an additional density experiment with stained MoDCs were carried out.

Figure 1. THP-1 cells plated at a density of 5x10⁴ cells/well (A) or 2.5x10⁴ cells/well (B). Imaged with transmitted light in imageXpress.

The nuclei, stained with Hoechst stain, were detected using DAPI filter in the microscope.

Shown in figure 2 is the loss of cells after staining were the cell loss increases with each staining step. Each time a supernatant is discarded after a wash, the cell density decreased.

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The cells in figure 2B and 2C have been stained with both Hoechst and membrane staining.

With each required wash, more initially plated cells are necessary to maintain a detectable amount of cells.

Figure 2. MoDCs are plated in untreated wells. A) MoDCs plated at a density of 2.5x10⁴ cells/well. The cells were stained with Hoechst nuclei staining and therefore the supernatant was discarded after centrifugation twice.

B) MoDCs plated at a density of 5x10⁴ cells/well. C) MoDCs plated at a density of 1x10⁵ cells/well. In B and C, the cells were stained with Hoechst and membrane staining, therefore the supernatant was discarded after centrifugation three times. The images do not show the membrane stain but only the Hoechst stain.

3.2 Immobilisation with coatings

To be able to perform live cell imaging for several hours and to follow the path of the antigen- adjuvant conjugate in single cells the MoDCs must be adherent to the bottom surface of the wells and not move out of the image area by time. Since immature MoDCs are non-adherent, three different coatings were investigated for the immobilisation of cells, collagen type 1, fibronectin and matrigel.

To evaluate the different coatings ability to immobilise cells, live cell imaging was used.

MoDCs were first plated on the different coatings for two hours before staining to let the cells have time to adhere to the coating. After the staining, cells were imaged every hour for a duration of 12-15 hours with the imageXpress microscope with a 20x or a 40x objective lens.

The images were processed with ImageJ software version 1.53a.

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Figure 3. MoDCs stained with Hoechst and plated at A) 5 x 10⁴ cells/well on 0.5 μg/cm² collagen type I and B) 1 x 10⁵ cells/well on 0.5 μg/cm² collagen type I. The arrow points at one immobilised cell.

Figure 4. MoDCs stained with Hoechst and plated at A) 5 x 10⁴ cells/well on 0.1 mg/ml matrigel and B) 1 x 10⁵ cells/well on 4 mg/ml matrigel. The arrow points at one immobilised cell.

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Figure 5. MoDCs stained with Hoechst and plated at 1 x 10⁵ cells/well on 0.5 μg/cm² (A) and 10 μg/cm²(B) fibronectin. The arrow points at one immobilised cell.

Collagen type I immobilises multiple cells at the lowest concentration used in the

immobilisation assay (figure. 3A). When an increased amount of cells were used on collagen coating, the immobilisation of the cells were impaired (figure. 3B). Matrigel do not

immobilise multiple cells at the lower concentrations (figure. 4A) but at higher concentrations it is possible to follow numerous of cells (figure. 4B). Immobilisation by using fibronectin and matrigel is not impaired at an increased cell density. Fibronectin, like matrigel, is concentration-dependent in the ability to immobilise cells (figure. 5).

For concentrations of matrigel below 4 mg/ml, an incomplete gel was formed. The incomplete gel got detached from the wells in the washing step. However, a thin, adhesive, layer

remained in the wells. Higher concentrations formed a stable gel that remained in the wells and disturbed the imaging in imageXpress and are therefore not presented.

In the experiment with 5 x 10⁴cells per well the cells were plated in 100 μl of cell culture media. The wells dried out between 6-7 hours of imaging. In the experiment with 1 x 10⁵cells per well the cells were plated in 200 μl of cell culture media and the wells dried out between 10-11 hours of imaging but the cell were out of focus after five hours.

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3.3 Activation from coatings

When introducing the antigen-adjuvant conjugate the MoDCs must be immature to both mimic the in vivo environment and because the internalisation of the complex is more efficient in immature cells. Because of this, it is important to investigate if the different coatings induce activation in MoDCs.

The cells were plated on the coatings for 15 hours to determine if the coating activated the cells. After 15 hours the plate was centrifuged and the supernatants were saved for an ELISA IL-12 (p40) assay. The cells left on the plate were analysed with flow cytometry. The

activation from the coatings was compared to a positive control (cells stimulated with LPS (E.

coli 0111: B4, Sigma) for the same duration as stimulation with coatings) and a negative control (cells without additional treatment).

Figure 6. Concentrations of IL-12 (p40) in the supernatant after 15 hours of stimulation on the different coatings.

The concentrations used is shown in table 1 and in the figure the concentrations are the highest furthest to the left of each coating and decreases to the right. Dunn’s test for Post Hoc analysis were used to determine if each pooled coating and the controls were significantly different P<0.01**.

Cells plated on collagen have increased secretion of IL-12 than cells plated on fibronectin and matrigel. However, collagen do not activate the cells as much as LPS treatment does. Figure 7 indicate that activation induced by fibronectin is concentration-dependent. However, the ELISA results imply the opposite (figure. 6). Collagen type 1 activates the cells at the lowest

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concentration used since the cells have formed clusters (figure. 7). Matrigel at the highest concentration that did not form a solid gel did not activate the cells as the round morphology is maintained after 15 h.

For the statistical analysis all concentrations from each coating were pooled to make larger groups. Since the sample size were still small after the pooling non-parametric tests were used. Dunn’s Post Hoc test showed that there is no statistically significant difference between the negative control and any of the coatings. However, matrigel is significantly different from the positive control unlike collagen and fibronectin (figure. 6).

Figure 7. Images of cells analysed with flow cytometry and ELISA after 15 h stimulation on coatings. The images were taken with an inverted microscope. Elongated and clustered cells are activated cells. Round cells are immature. MoDCs plated on 10 μg/cm² fibronectin (A), 0.5 μg/cm²fibronectin (B), 0.5 μg/cm²collagen type 1(C), 4 mg/ml matrigel (D). LPS treated MoDCs as positive control (E) and MoDCs any treatment as negative control (F).

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Figure 8. Mean fluorescence intensity (MFI) of each coating for the three activation markers, HLA-DR, CD83 and CD86. The flow cytometry analysis was performed on MoDCs after 15 hours of stimulation on the different coatings. The concentrations used is shown in table 2 and in the figure the concentrations are the highest furthest to the left of each coating and decreases to the right.

HLA-DR, CD86 and CD83 are receptors that are upregulated on activated MoDCs (23,24).

For the flow cytometry each coating and concentration had a LPS treated control. Fibronectin and matrigel have a lower expression of HLA-DR and CD83 than the LPS treated controls (figure. 8). Collagen had the same, or higher expression of the same receptors as the LPS treated control. There were fewer than 1x10⁴ cells detected in the majority of the treatments which means that the result is unreliable.

3.4 Membrane staining

By staining the surface receptor HLA-A2 it is possible to distinguish intra- and extracellular parts. Illustrated in figure 9 is the possibility to merge different fluorescence channels. By using merged channels, it is possible to localise the peptide and antibody.

Figure 9. THP-1 cells stained with Hoechst nuclei staining and membrane staining. A) The FITC channel (HLA- A2 receptor) of the image displayed i.e. only the membrane is visible. B) Both FITC and DAPI (nuclei) channel of the image displayed, both the membrane and the nuclei is visible.

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4. Discussion

The initial density of MoDCs depend on the number of washing steps needed for the staining.

For two washing steps 1 x 10⁵ cells per well is the optimal density of the ones investigated in this study. However, the number of cells varies a lot from well to well and for each time experiment is performed. To plate more MoDCs in a separate plate for the staining, then count the cells and plate equal amount of cells in each well on the coated plate for live cell imaging might be a solution for the variating amount of cells in the wells. This might also reduce the amount of activated cells plated for live cell imaging since the cells that have been activated due to handling in the staining process would have adhered to the first plate and not been transferred to the final plate. If the staining would occur in a separate plate, 25 x 10⁴ cells per well would be a cell density to move further on with.

Collagen and fibronectin seem to be concentrations-dependent in both immobilisation and activation of cells. Matrigel do not induce activation even in high concentrations but is concentration-dependent in the ability to immobilise cells. The correlation between the activation and immobilisation from collagen and fibronectin raises the question: is the immobilised cells in collagen and fibronectin images activated and therefore naturally adherent? When 1 x 10⁵cells were plated in collagen, a majority of all the cells were not immobilised. This was seen in all wells coated with collagen but not in any of the other coatings. The efficiency of the binding of cells to collagen might be dependent on the cell density. A higher cell density might reduce the efficiency of collagen binding. To investigate this further an assay with different cell densities plated on collagen could be performed.

IL-12 secretion induced by activation from the different types of coatings only showed

statistical significance between the matrigel and the positive control. As mentioned before the activation of the cells is concentration-dependent for fibronectin and collagen, therefore it would be interesting to be able to do statistical analysis within the group of each coating. To enable this comparison, multiple ELISA assays on supernatants from either the same

experiment of repeated experiments must be performed to obtain more data. However, this is not a necessity for this study since matrigel at a concentration between 1-4 mg/ml has been shown to be an adequate coating for the purpose.

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By staining a surface receptor, it is possible to determine if the peptide and antibody are intra- or extracellular by merging the channels of the different staining. This is an essential part in the recognition of where the peptide is released from the antibody. It is also important that the channels can remain unmerged to be able to measure the fluorescence of one channel. By using both merged and unmerged channel, it should be possible to localise and quantify the peptide and antibody. An experiment investigating the possibility of localisation with the actual antigen-adjuvant conjugate is yet to be performed. It is therefore difficult to conclude that the developed assay allows determination of the localisation of antigen-adjuvant

conjugate and peptide release.

The assay developed in this study aimed to investigate the internalisation of an antigen- adjuvant conjugate, similar to a part of the study performed by Rosalia et al. (25), that investigated the internalisation of synthetic long peptides by using a confocal microscope. In contrast to the assay developed in this study, Rosalia et al incubated fluorescently labelled peptides with DCs for 2,5 h and 24 h and washed cells after the incubation to remove excess and unbound peptide. The results from this study showed that the peptides are internalised within two hours and the uptake continues during the 24 h incubation. The assay developed in the present study would provide a much more detailed description of the internalisation process since more time points can be analysed and the whole process can be followed in the same cell. Both Reuter et al. (9) and Chatterjee et al. (3) used antigens fused with antibodies that target DC surface receptors and investigated the internalisation and cross-presentation of these conjugates. Both studies demonstrated that CD40 receptors are internalised in early endosomes. Reuter et al. also reported that the time to maximum internalisation of CD40 receptors are 30 minutes. This gives more motive to investigate the early time points of the internalisation.

4.1 Limitation of the present study

The cell culture media evaporated during imaging. This is probably due to that the cells were imaged in 37°C and 5% CO2 without the lid on. Even if it is possible to image the cells for ten hours when 200 μl cell culture media is used, the volume of media decreases with time which increases the cell concentration and changes the condition for the cells. This might affect the health of the cells and thus the result of the experiment. The cells were also out of focus after five hours of imaging which is a problem since this obstruct the ability to follow the

internalisation for eight hours. These two problems were not further investigated due to time

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restrictions. The same imaging experiment should be performed to investigate if the

unfocused images are a recurring problem. A way to prevent the evaporation of cell culture media might be to use a gas permeable sealing film. This would prevent the media from evaporating but at the same time provide an adequate gas exchange to keep the favourable environment. It is however important to investigate these films effect on the quality of the images. Because the developed assay does not enable imaging for more than five hours the time aspect of the internalisation might not be possible to determine. Five hours might be enough time to answer the question of when the antigen-adjuvant conjugate is internalised and when peptide release occur. To know this, the assay must be performed with the antigen- adjuvant conjugate. For now, the time aspect of the aim is not fulfilled.

The data from the flow cytometry analysis is unreliable since there were very few cells in each sample. The low amount of cells can be a consequence of discarding supernatant

throughout the staining. When there are few cells, expression of a certain receptor on one cell makes a large difference in the data and can give misleading results.

The statistical analysis was performed on only one ELISA assay and the result might not be accurate due to the small groups of data. More data must be obtained for more accurate statistics.

4.2 Future perspectives

Future studies must initially develop the assay further. The cause of the unfocused images must be investigated and solved to ensure the quality of the images. A method to prevent evaporation of the cell culture media during imaging but still provide adequate gas exchange needs to be explored. When an assay with the possibility to study the time aspect of the internalisation and peptide release as well as the localisation of the peptide release is developed, the antigen-adjuvant conjugate can be introduced to MoDCs and the internalisation and peptide release can be studied.

To investigate the internalisation by using live cell imaging, an excess of the antigen-adjuvant conjugate will be incubating during imaging. Since both antibody and peptide will be

fluorescently labelled for the internalisation study, this might obscure the imaging. The noise in the images might prevent quantification of internalised antigen-adjuvant conjugate. Most studies that have imaged fluorescently labelled components wash away excess and unbound

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fluorescent materials (3,25–27). If obscured imaging occurs, another approach might be necessary. This approach could be more similar to the assay used by Rosalia et al. (25). The antigen-adjuvant conjugate would be incubated with MoDCs for different time points during eight hours and then excess and unbound conjugate would be washed away. More time points the first few hours would be used to obtain more detailed data of the internalisation. When a more detailed data of the internalisation is obtained, the antigen-adjuvant complex can be further developed to be optimal from both an effect and safety point of view.

4.3 Concluding remarks

To conclude, the assay must be further developed for the aim to be completely fulfilled.

Firstly, the method suggested to ensure a constant amount of cells in all wells must be implemented. Secondly, an assay that provides the possibility to image cells with continuous quality for eight hours must be developed. Lastly, the developed assay must be evaluated by preforming an experiment were the antigen-adjuvant conjugate is introduced to MoDCs. The use of matrigel at a concentration between 1-4 mg/ml can be concluded as the optimal coating. Matrigel at these concentrations does not induce activation of the cells and immobilise the cells adequately which enable detection of single cells for several hours.

Acknowledgements

First and foremost, I would like to thank my supervisor, Ida Olsson, who has been an

invaluable help in all aspects of this project. Thank you for your guidance and encouragement to always learn new thing throughout the entire semester. I would also like to thank everyone in the Mangsbo group for all the help with both theoretical and practical questions and issues.

A special thanks to Sara Mangsbo for the opportunity to do my project in the exiting and interesting field of immune oncology. Lastly, I would like to thank Jamie Morrison (Division for Protein Drug Design, Uppsala University) for providing me with the different coatings and helping me with the protocols.

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Appendix

Appendix 1. MoDCs stained with Hoechst nuclei staining. Plated at the density 5x10⁴ cells/well. Three images from different time points of each concentration of a coating is shown. The arrow in each image points at one cell that can be tracked for the whole timespan.

The images are taken with a 20x objective lens.

0 h 2 h 4 h

Media controlCollagen type I 10 μg/cm2 5 μg/cm2 1 μg/cm2 0.5 μg/cm2

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0 h 2 h 4 h

Fibronectin 10 μg/cm2 5 μg/cm2 1 μg/cm2 0.5 μg/cm2

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0 h 2 h 4 h

Matrigel 1 mg/ml 0.75 mg/ml 0.5 mg/ml 0.1 mg/ml

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Appendix 4. MoDCs stained with Hoechst nuclei staining. Plated at the density 1x10⁵ cells/well. Three images from different time points of each concentration of a coating is shown. The arrow in each image points at one cell that can be tracked for the whole timespan.

0 h 2 h 4 h

Media controlCollagen type I 5 μg/cm2 1 μg/cm2 0.5 μg/cm2

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Appendix 5. MoDCs stained with Hoechst nuclei staining. Plated at the density 1x10⁵ cells/well. Three images from different time points of each concentration of a coating is shown. The arrow in each image points at one cell that can be tracked for the whole timespan.

0 h 2 h 4 h

Fibronectin 10 μg/cm2 5 μg/cm2 1 μg/cm2 0.5 μg/cm2

Matrigel 4 mg/ml

Internalisation of antigen-adjuvant conjugate in human dendritic cells: An assay development for using live cell imaging