Enzyme-Linked Immuno Spot (ELISpot) is a technique which quantifies rare immune cells that release biomarkers such as cytokines in response to antigenic stimulation. ELISpot is a highly sensitive method to test immune modulators, novel vaccine candidates or de-risk immunogenicity testing in an antigen-specific CD4 and/or CD8 T cell assay.
| Condition | Aim |
| No Stimulation | Negative control |
| PMA | Positive control |
| CERI (CMV, EBV, RSV, Influenza) | MHC-I restricted peptide pool to evaluate modulation of CD8+ T-cell memory response |
| CPI (CMV, Parainfluenza, Influenza) | Positive protein antigens to evaluate modulation of CD4+ T-cell memory response |
| CEF (CMV, EBV, Influenza) | MHC-I restricted peptide pool to evaluate modulation of CD8+ T-cell memory response |
| Cyclosporin A (CsA) | Inhibition of immune response |
Spot Forming Units (SPU) for IFN-γ per 100,000 PBMC from CERI, CPI and CEF antigens for three donors
Therapeutic modulation of TH17 CD4+ T cell differentiation or effector function
T helper 17 (Th17) cells, are a subset of CD4 T cells distinguished by their production of IL-17. While crucial for protecting the body against extracellular pathogens, Th17 cells have also been implicated in autoimmune disease. Discovering therapies that target either the differentiation or effector function of Th17 cells could therefore prove beneficial for the treatment of some autoimmune diseases.
Evaluation of therapeutic modulation of Th17 differentiation
Figure 1: Polarisation of Th17 cells. Naïve CD4 cells were cultured under Th17 polarising conditions for 12 days in the presence or absence of Ursolic Acid. CD4 T cells were assessed for proliferation by CTV dilution; intracellular cytokine staining (ICS) of IL-17 and IL-10 by flow cytometry. On Day 12, Supernatants were collected and evaluated for IL-17 and IL-10 levels by MSD. RoRγT inhibitor Ursolic Acid showed selective inhibition of IL-17 production by intracellular cytokine staining (ICS) and MSD.
Evaluation of therapeutic modulation of Th17 effector function
Figure 2: Inhibition of Th17 cell effector function. Magnetically sorted memory Th17 cells were polyclonally stimulated under Th17 conditions in the presence or absence of ursolic acid or FK506 for 5 days. A percentage of Th17 cells are polyfunctional (IL- 17A+IFNg+) and refractory to inhibition by ursolic acid (an inhibitor of Th17 differentiation) but not FK506.
Evaluation of therapeutic modulation of antigen-specific memory T cell responses to recall antigens
I. T cell response to Tetanus Toxoid, Influenza and PPD antigens
II. Dose response to Influenza antigen
Antigen specific T cells response to a recall antigens. (I) Healthy donors PBMC were stimulated with PHA-M or triple antigen cocktail (Tetanus Toxoid, Influenza and PPD). Cyclosporin was used as a reference treatment. (II) Dose response to Influenza antigen. CD4 and CD8 T cells proliferation was measured by flow cytometry using CTV dilutions.
Enhance T cell effector function: benchmark novel therapies against immune checkpoint blocker (anti-PD-1) in a 1-way MLR
PD-1 blockade does not enhance CD4 T cell proliferation in a 1-way MLR as determined by dye dilution of CTV labelled CD4 T cell using flow cytometry.
3D Tumour Killing Models: Tools for screening immune or tumour targeted therapeutics
Figure 1: SKOV-3 NLR tumour cells were seeded into 96 well plates, PBMC were added once spheroids were established in the presence of Pembrolizumab or IgG4 control or vehicle (untreated) and imaged every 4 hours for 96 hours using the CellCyte X. Relative spheroid area (%) was measured (A). AUC statistics were calculated using GraphPad Prism v9.5.0
Enzyme-Linked Immuno Spot (ELISpot) is a technique which quantifies immune cells of low abundance which release biomarkers such as cytokines in response to antigenic stimulation. ELISpot is a highly sensitive method to test immune modulators, novel vaccine candidates or de-risk immunogenicity testing in an antigen-specific CD4 and/or CD8 T cell assay.
| Condition | Aim |
| No Stimulation | Negative control |
| PMA | Positive control |
| CERI (CMV, EBV, RSV, Influenza) | MHC-I restricted peptide pool to evaluate modulation of CD8+ T-cell memory response |
| CPI (CMV, Parainfluenza, Influenza) | Positive protein antigens to evaluate modulation of CD4+ T-cell memory response |
| CEF (CMV, EBV, Influenza) | MHC-I restricted peptide pool to evaluate modulation of CD8+ T-cell memory response |
| Cyclosporin A (CsA) | Inhibition of immune response |
Spot Forming Units (SPU) for IFN-γ per 100,000 PBMC from CERI, CPI and CEF antigens for three donors
Evaluation of therapeutic modulation of antigen-specific memory T cell responses to recall antigens
I. T cell response to Tetanus Toxoid, Influenza and PPD antigens
II. Dose response to Influenza antigen
Antigen specific T cells response to a recall antigens. (I) Healthy donors PBMC were stimulated with PHA-M or triple antigen cocktail (Tetanus Toxoid, Influenza and PPD). Cyclosporin was used as a reference treatment. (II) Dose response to Influenza antigen. CD4 and CD8 T cells proliferation was measured by flow cytometry using CTV dilutions.
Dendritic cells fed with tumour cell lysates prime rare neoantigen-specific CD4 T cells and cross present antigen to prime neoantigen-specific naive CD8 T cells
Therapies targeting the modification of tumor cells to increase neoantigen expression and improve immune cell recognition are currently under development. Detecting these subtle changes typically involves several cycles of dendritic cell restimulation of T cells, followed by the sensitive measurement of rare neoantigen-specific T cell responses using IFNγ ELISPOT assays. The schematic below shows the assay design with multiple round of dendritic cell (DC) restimulation to expand rare neoantigen T cell responses.
IFNγ release from activated tumour neoantigen-specific CD4 T cells
IFNγ release from activated tumour neoantigen-specific naive CD8 T cells
Figures: CD4 or naïve CD8 T cells were co-cultured with unloaded dendritic cells (DC), or with DC loaded with tumour cells that had been previously treated with media or vehicle. Three rounds of stimulation of T cells with DC were performed (* single round of DC stimulation). PMA and ionomycin were added as positive controls during the final round of stimulation. T cell stimulation was assessed by measuring the number of spot forming units (SFU) by IFNγ ELISpot. Media and T cell only wells were included as background controls. Number of SFU per 100,000 plated T cells, with mean of two (experimental controls) or three (all others) replicates ± SEM shown. Representative images of CD4 and naïve CD8 T cell IFNγELISpot. Each blue dot represents one IFNγ-producing T cell. ULOD = Upper Limit of Detection
Tonsillar re-aggregate model
Tonsils are a secondary lymphoid tissue rich in B cell subsets and the specialized, anatomically restricted T follicular helper (Tfh) subset of CD4 T cells. Tonsils provide an accessible source of cells for those interested in B cell immunology, Tfh biology and vaccine development. The tonsillar cell re-aggregate model provides functional insight into human germinal center biology, allowing measurement of key features including naive B cell differentiation, plasma cell/plasmablast differentiation, and antibody production.
