Immunology

Immunology explores our immune system, a complex and structured network of molecular and cellular components that exploits our immunity, the front-line of defence against external molecules and pathogens.

The immune system is composed of innate and adaptive immunity. The former is non-specific but continuously tackles all potential pathogens that enter our body. These mechanisms protect our body and fight any infection in the first instance. If the innate immunity defence mechanism is unable to clear off the pathogen(s) from our body, the adaptive immunity acts as the second line of defence and gets activated. This second line of defence includes recruitment of two different groups of specialised cells: B and T lymphocytes.  B lymphocytes are known to produce antibodies, molecules that can target specific pathogens, especially those that circulate freely in the bloodstream. Activated T lymphocytes will chase bacteria and viruses that have colonised our tissues and will remove infected cells and de facto, the pathogen(s). The adaptive immunity also maintains the memory of encountered infections, enabling it to act swiftly and effectively if it recurs. The mechanistic action of vaccines is based on harnessing this aspect of our innate and adaptive immunity. The objective here is to prime the body allowing it to respond rapidly following exposure to a pathogen, thereby destroying it without causing the disease.

Sepsis is another potential life-threatening condition usually triggered by microbial infection. This leads to dysregulated immune system resulting in activation of systemic immune responses and hyper activation of immune cells producing pro-inflammatory cytokines and chemokines.  

Similarly, some viruses (like SARS/MERS coronaviruses or influenza) could drive our immune system to over-reactive state resulting in a systemic failure called “cytokine storm”. Cytokines are small molecules released by cells, including immune cells, used to activate our body’s response against infection. However, like in the Covid-19 pandemic, the SARS-CoV-2 virus infection leads to a hyperactive and impaired immune system resulting in a state of excessive inflammation. This could be of serious consequences and even fatal.

Cellomatics Biosciences Ltd. offers well validated preclinical models to investigate the role of various Immune cells in regulating key signalling oathways in inflammatory diseases and cancer. In-house, we have the capabilities to recruit healthy volunteers, collect and isolate fresh immune cells for downstream assays. These models are suitable for low to high throughput studies.

Adhesion

Adhesion molecules are cell surface proteins that mediate the interaction between cells, or between cells and the extracellular matrix (ECM). Cell adhesion plays an important regulatory role in cell mediated immunity.

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Leukocyte adhesion using HUVEC co-culture

A statistically significant increase in leukocyte adhesion was observed on TNFα stimulated HUVEC monolayers when compared to HUVEC (untreated), HUVEC+ leukocyte (untreated) and IC87114 treatment (PI3K Inhibitor) (*p<0.05; **p<0.01; ***p<0.001±SEM).

Migration

Immune cells play an important regulatory role in immune homeostasis. To support this process, migration of immune cells is critical for the delivery of protective immune responses to tissues.

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Chemotaxis

Neutrophil migration through the HUVEC monolayer

Treatment with TNFa significanty increased fMLP-induced neutrophil migration through HUVEC monolayers (ns=not significant; **p<0.01; ***p<0.001±SEM). fMLP is a chemoattractant.

Chemokinesis

A scratch was formed on a A549 cell monolayer and treated with Olaparib (Ola), 5-Fluorouracil (5-FU), or a combination of both. Images were acquired using a live imaging platform (DETAILS) at the indicated time points (hrs). The wound size/scratch was measured at all time points. The wound closure percentage was analysed assuming 0% closure at 0 hours, and calculating all the following time points as ratio to 0 hours [ *p<0.05; **p<0.01;***p<0.001; ± SEM].

FACS Based Analysis

Flow cytometry has various research applications including immunophenotyping, measuring intracellular cytokine production, cellular proliferation, assessing cell viability and analysis of cell cycle, rare events, stem cells and fluorescent proteins.

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Neutrophil characterisation

Neutrophils were isolated from fresh peripheral blood using a commercially available isolation kit (details) and the purity of cells was subsequently analysed by FACS for neutrophil surface markers (CD45 and CD66b). Figures A 1-4 represent the gating strategy employed for characterising the neutrophils. The neutrophil population was subsequently stimulated with [concentration] TNFα for 4 hours and an increase in cells expressing the LFA-1 surface antigen was observed (B1, 2).  

T-cell subpopulation characterisation: CD4+, CD27+, CD28+

T-cell lymphocytes were isolated from peripheral blood using commercially available isolation kits. To characterise the subpopulation of CD4+T-cells, the lymphocytes were stained for surface markers (CD4, CD27, CD28) along with viability dye and analysed by FACS.  The CD4+T cells were further sub-characterised and 82.13% cells were positive for CD4, CD27 and CD28. The expression of these co-stimulatory markers are helpful in investigating the immune responses in young and old donors.

Regulatory T (TReg)-Cell Characterisation: CD4+CD25+CD127dim/-

Regulatory T cells (T Reg) were isolated from peripheral blood using commercially available isolation kits. 

The cells were labelled with CD4, CD25 and CD127. It was observed that 45.57% of the cells were CD4+CD25+; within this a sub-population of 92.20% of the CD4+CD25+ cells expressed low levels of the CD127 surface antigen.

THP-1 characterisation after PMA-differentiation

Stimulation of THP-1 cells (human monocyte model) with PMA induces differentiation and expression of a macrophage-like phenotype. The PMA-differentiated THP-1 cells were stained with macrophage surface markers (CD11; CD40) and analysed using FACS. Figures A and B demonstrate that the expression of CD11 and CD40 was higher following PMA-induced differentiation of THP-1 cells (red) when compared to undifferentiated cells (green).

Differentiation of primary monocytes to M0, M1 and M2 MΦ

CD14+ monocytes were isolated from PBMCs using a commercially available kit. Monocyte differentiation to M0 macrophages (Panel A) was induced and inhibited by M-CSF and SB203580, respectively. Polarisation of M0 macrophages to M1 macrophages (Panel B) was induced by M1 cytokine cocktail and inhibited following treatment with Dexamethasone. Polarisation of M0 macrophages to M2 macrophages (Panel C) was induced by M2 cytokine cocktail and inhibited following treatment with Tofacitinib. Characterisation of M0, M1 and M2 macrophages under experimental conditions was performed by flow cytometric analysis of the expression of cell suface markers (CD86, CD80 and CD206).

PBMCs Based Assays

Peripheral blood mononuclear cells (PBMC) provide selective responses to the immune system and are the major immune cells in the human body. PBMCs include lymphocytes (T cells, B cells, and NK cells), monocytes, and dendritic cells. PBMCs based assays explore the role of these cells in immune modulation, cell proliferation/cytotoxicity and their role in targeting specific cancer cells.

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PBMCs – anti-CD3 stimulation

Freshly isolated Peripheral Blood Mononuclear cells (PBMCs) were seeded onto anti-CD3 coated U-bottom plates and further stimulated with soluble anti-CD28 for 48 hours. PBMCs were also treated with Cyclosporin A prior to stimulation. Isotype of anti-CD3 was used as a background control. Supernatants were analysed for inflammatory markers using a Luminex Multiplex Assay (n=3±SEM; *p<0.05; ****p<0.0001).

PBMCs – anti-CD3/anti-CD28 stimulation

Freshly isolated Peripheral Blood Mononuclear cells (PBMCs) were seeded onto anti-CD3 coated U-bottom plates and further stimulated with soluble anti-CD28 for 48 hours. PBMCs were also treated with Cyclosporin A prior to stimulation. Isotype of anti-CD3 was used as a background control. Supernatants were analysed for inflammatory markers using Luminex Multiplex Assay (n=3±SEM; *p<0.05; ****p<0.0001).

PBMCs – anti-CD3/anti-CD28 stimulation for 72 hours

Freshly isolated Peripheral Blood Mononuclear cells (PBMCs) were seeded onto anti-CD3 coated U-bottom plates and further stimulated with soluble anti-CD28 for 24, 48 and 72 hours. PBMCs were also treated with Cyclosporin prior to stimulation. Isotype of anti-CD3 was used as a background control. Supernatants were analysed for inflammatory markers using Luminex Multiplex Assay (n=5±SEM; *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001).

PBMCs – anti-CD3 stimulation

Effect of ICAM-1 inhibitor on PBMC proliferation.

The absorbance (570 nM) for PHA stimulated PBMC cells was significantly higher when compared to the Vehicle Control (unstimulated) (##p<0.01). A statistically significant reduction of absorbance was observed for PBMC (PHA stimulated) treated with ICAM-1 inhibitor when compared to PHA stimulated control. Differences were identified using a one-way ANOVA followed by a Dunnett’s post-hoc multiple comparison test (*p<0.05; **p<0.01; ***p<0.001; ±SEM).

Effect of ICAM-1 inhibitor on release of inflammatory marker levels from PBMC.

PHA stimulation of PBMCs significantly increased the expression levels of inflammatory markers (e.g. TNFa) when compared to Vehicle Control (###p<0.001). A dose-dependent reduction in the release of the markers was observed for PBMCs treated with ICAM-1 inhibitor when compared to PHA stimulated PBMCs. Analysed using one-way ANOVA followed by Sidak’s post-hoc multiple comparison test (**p<0.01; ***p<0.001; ±SEM).

PBMCs proliferate in response to anti-CD3 stimulation

PBMCs from 2 different healthy volunteers were stimulated with increasing concentrations of anti-CD3 antibody (0 µM, 0.25 µM, 1 µM and 5 µM) over the period of 144 hours (6 days) and the cell proliferation was recorded. A dose dependent increase in PBMC cell counts was observed after 48 hours of stimulation (n=5±SEM).

T-Cells based assays

T cells coordinate multiple aspects of adaptive immunity throughout life, including responses to pathogens, allergens, and tumours. T cells control multiple insults simultaneously throughout the body and maintain immune homeostasis over decades.

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Naïve CD4+ T-cells polarised to T Helper (Th)-2 cells

Freshly isolate naïve CD4+ T-cells were polarized to Th2 cells by stimulating with anti CD3 for 7-10 days. IL4, IL5 and IFNγ levels were then measure in cell supernatants. Results are expressed as mean ± SEM (n=5)

Naïve CD4+ T-cells stimulated with Derp1

Naïve CD4+ T-cells were isolated from the peripheral blood of an Atopic donor and were stimulated with Derp1 for 5 days. Levels of inflammatory mediators including IL6, IL17 and TNFα were measured in cell supernatants by multiplex immunoassay. Derp1 stimulation induced a statistically significant increase in the levels of the inflammatory mediators (one-way Anova **p<0.01; mean ± SEM).

T-cell Proliferation

CD4+ T-cells were seeded in U-bottom 96-well plates. Cells were placed under different treatment regimes for 8 days. Cell counts were taken at days 1, 2, 3,6 and 8. Cells were subjected to the following treatments: unstimulated, stimulated (CD3 & CD28), and stimulated + inhibitor (TGFβ1).  Average cell counts were plotted using GraphPad Prism. Cell viability was analysed for each timepoint using CellTitre-Fluor™ (n=2, mean±SEM).

B Lymphocyte Based Assays

In addition to their role in humoral immunity, it is now established that B lymphocytes contribute directly to cellular immunity via at least three mechanisms: (1) they serve as antigen-presenting cells (APCs) that enhance T lymphocyte–mediated immunity; (2) they function as bona fide cellular effectors that produce inflammatory cytokines; and (3) modulate immune responses.

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Pan-B lymphocyte IgE release assay

Pan B lymphocytes were freshly isolated from the peripheral blood of a healthy volunteer. Cells were stimulated with IL4+IL13 for 48 hours in the presence or absence of Dexamethasone. Increased IgE release was observed following IL4+IL13 stimulation; this was inhibited by pre-treatment with Dexamethasone (mean ± SEM). 

Macrophage Based Assays

Macrophages are effector cells of the innate immune system that phagocytose bacteria and secrete both pro-inflammatory and antimicrobial mediators. In addition, macrophages play an important role in eliminating diseased and damaged cells through their programmed cell death. They regulate lymphocyte activation and proliferation, and they are essential in the activation process of T- and B-lymphocytes by antigens and allogenic cells. Enhanced bactericidal activity of “activated macrophages” is based on immunologically linked mechanisms involving lymphocytes.

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Difference in phagocytic potential of M1 and M2 MΦ

Primary monocytes (CD14+) were isolated from PBMCs of healthy donors and were treated with M-CSF for 6 days. On day 6, the naïve macrophages were exposed to GM-CSF + IFNγ and M-CSF to generate M1 and M2 macrophages (MΦ) respectively. The cells were then cultured with fluorescent zymosan particles for 3 h (green-phagocytic marker). After 3 hours, the cells were washed and thenstained with a fluorescent membrane marker (red) and DAPI (blue-nucleus).

The images above showed M2 macrophages engulfed higher numbers of zymosan particles when compared to M1 macrophages (Objective= 20X).

Difference in phagocytic potential of M1 and M2 MΦ

The fluorescent images from the phagocytosis assays were analysed using ImageJ. Each dot on the scatter plot represents 1 image field at 20X objective.

[A] Percentage of Zymosan positive cells =  (Cells with zymosan particles/ Total number of cells per field)*100; [B] Phagocytic Index = (Average number of particles per positive cell)/ Percentage of Zymosan positive cells

M2 Macrophages (n=9) showed higher phagocytic index when compared to M1 macrophages (n=5) [±SEM].

Phagocytosis assay using PMA-differentiated THP-1 cells

THP-1 cells (immortalised human monocyte model) were treated with PMA to induce macrophage like characteristics. Phagocytosis was subsequently assessed by incubating cells with fluorescent zymosan particles (green). The images were captured at 20x objective and analysed for phagocytic index using Image J (n=5±SEM).

Differentiation of primary monocytes to M0, M1 and M2 MΦ

CD14+ monocytes were isolated from PBMCs for macrophage (MΦ) differentiation. Monocyte differentiation to M0 MΦ (Panel A) was induced and inhibited following treatment with M-CSF and SB203580, respectively. Polarisation of M0 to M1 MΦ (Panel B) was induced and inhibited following treatment with LPS and Dexamethasone, respectively. Polarisation of M0 to M2 MΦ (Panel C) was induced and inhibited following treatment with IL-4/IL-10 cocktail and Tofacitinib, respectively. Characterisation of M0, M1 and M2 MΦ in the various conditions was analysed through expression levels of cell surface CD86, CD80 and CD206 markers using flow cytometry.

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