CD4+ T cell

Immune-Oncology Assays

  • Polyclonal
  • MLR
  • Antigen specificity
  • nTreg suppression assay
  • iTreg polarisation assay

Autoimmunity Assays

  • Th1/2/17 polarisation
  • Th17 function
  • Tr1/iTreg polarisation
  • nTreg suppression
  • Tfh, naïve
  • CM and EM phenotype/function
  • Polyclonal T cell proliferation
  • Cytokine release

Inflammation Assays

  • Cytokine release

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.

ConditionAim
No StimulationNegative control
PMAPositive 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
Figure 1: The ELISpot visual image results showing four donors under four conditions: Unstimulated (negative control), stimulated with PMA (positive control), stimulated with CERI, and stimulated with CPI antigens. Each purple spot represents one IFN-γ producing cell.

Spot Forming Units (SPU) for IFN-γ per 100,000 PBMC from CERI, CPI and CEF antigens for three donors

Figure 2: T cell responses from three donors using stimuli, CERI (I), CPI (II) and CEF (III).  Expressed as spot forming units per 100,000 donor PBMC. Depletion of CD4 or CD8 T cells demonstrates specificity of response to peptide pools. Each value represents the mean of triplicate wells ±SEM.

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. Ursolic Acid showed selective inhibition of IL-17 production by ICS and MSD.

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

Schematic description of 1-way MLR: purified CD14 cells differentiated into DC +/- LPS maturation co-cultured with purified allogenic CTV labelled CD4 T cells.
PD-1 blockade increases IFN-γ production in a 1-way MLR. Levels of IFN-γ were measured by MSD in cell culture supernatants.


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.

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