ImmuneCarta

Defining the Response™

Functional Profiling

Overview

The functional state of an immune cell can be assessed by flow cytometry under normal and pathological conditions.

ImmuneCarta provides detailed information on the status of the immune system and provides experimental and analytical tools that link phenotypic profiles with functional markers of:

  • Proliferation
  • Cell cycling
  • Kinase phosphorylation
  • Cytokine secretion
  • Degranulation

Our comprehensive approach aims to identify signatures of immune protection and therapeutic response.

The functional profile of distinct immune cell subsets can be measured in whole blood, PBMC (peripheral blood mononuclear cells) and tissues using the following assays:

  • ICS (Intracellular Cytokine Staining)
    • Measure of cytokine production and degranulation
  • CFSE (Carboxyfluorescein succinimidyl ester)
    • Measure of proliferation
  • Phospho Flow Cytometry
    • Measure changes in the phosphorylation state of intracellular kinases in response to different activation/suppression signals
  • Cell Cycling
    • Determine the cells that are in the active phase of the cell cycle, using Ki-67 as a nuclear marker
  • BAT (Basophil Activation Test)
    • Measure the activation and degranulation of basophils in whole blood

ICS


ImmuneCarta provides detailed information on the immune status by combining ICS (Intracellular Cytokine Staining) with cell surface markers and HLA class I multimers to detect Antigen-Specific Responses.

After a short in vitro stimulation with proteins, peptides, or antigen-presenting cells, responsive subsets are analyzed within this cellular mixture to determine the distribution of functional subsets and ultimately signatures of immune competence.

Benefits and Applications

  • Obtain phenotypic and functional data in a single sample
  • Identify the frequency of reactive antigen-specific cells in various matrices
  • Determine precise phenotyping of the responding cell populations with a highly multiparametric read-out (up to 18 parameters)
  • Analyze the relative distribution of functional subsets through multiplexing functions

Immune response monitoring using ICS Intracellular Cytokine Staining Process to detect response to antigen
ICS Process to detect antigen specific immune responses

CFSE


ImmuneCarta offers a qualified CFSE (Carboxyfluorescein Succinimidyl Ester) Assay used to measure and characterize cellular proliferation in response to various stimulations. Additional cell surface and intracellular markers are combined with CFSE staining to assess responsiveness of specific cell subsets.

In addition to monitoring cellular proliferation, our CFSE allows us to determine the number of divisions that a cell has undergone in response to clinical treatment or in-vitro stimulations.

Benefits and Applications

  • Provides information on the functionality and responsiveness of cell subsets from diseased and healthy individuals, both before and after treatment
  • Identifies the cellular subsets that are actively proliferating, as well as the cytokines being produced at the single cell level, by combining with phenotypic and functional markers
  • Quantifies the number of cell divisions a specific subset has undergone (proliferation index) with our highly sensitive CFSE assay

Immune response monitoring using CFSE or Carboxyfluorescein Succinimidyl Ester Assay-analysis of T cell proliferation and cell division
CFSE analysis depicting a dot plot (A) of proliferating (CFSElow) CD8+ and CD8- T cells and a cell cycle analysis (B) showing the number of cellular divisions within the total T cell pool.

Phospho Flow Cytometry


Phospho Flow Cytometry is a rapidly developing flow cytometry technology that detects multiple intracellular signaling molecules in immune cells at the single-cell level. Abnormal kinase activity has been associated with disease, including cancer and autoimmunity.

ImmuneCarta uses multiparametric flow cytometry to analyze the phosphorylation state of targeted kinase(s), both in vitro and ex vivo, with and without stimulation. Application of our expertise to understand cell signaling alterations under normal and pathological conditions is critical for the development of novel kinase inhibitors for cancer and autoimmunity.

Benefits and Applications

  • Determine activation status of multiple signaling pathways in specific cellular subsets at the single cell level in several matrices: whole blood, PBMC (peripheral blood mononuclear cells), tissues
  • Multiplex multiple phosphorylated targets to gain insights to membrane proximal and distal signaling events in distinct cellular subsets

Immune response monitoring with Phospho flow cytometry to analyze cell types and kinase phosphorylation states

(A) Dot plot depicting the granularity and size of the different cellular subsets analyzed. (B) Histogram showing the intensity of phospho-p38 in the various subsets analyzed both at baseline and following PMA/Ionomycin stimulation

Ki67 in Cell Cycling


ImmuneCarta uses Ki67 as an early activation marker to identify cells which have entered into cell cycle.

Additional cell surface and intracellular markers are combined with Ki67 staining to identify the cell subset of interest along with other functional attributes. The nuclear antigen Ki67 is expressed in all active phases of the cell cycle (G1, S, G2 and M), but is absent from resting cells (G0).

Benefits and Applications

  • Identify cells that have entered into the cell cycle using Ki67 as an early activation marker.
  • Correlate the clinical course of cancer using Ki67 as a prognostic biomarker

Immune response monitoring using Ki-67 marker to identify cells in active phases
Schematic representation of the cell cycle.
Nuclear Ki-67 is expressed in all phases except G0 (Resting cell).

BAT


The Basophil Activation Test (BAT) assay is performed on whole blood and uses flow cytometry to measure CD63 expression, a degranulation marker, following basophil activation in vitro.

ImmuneCarta uses the Basophil Activation Test (BAT) to identify the fraction of HLA-DR-CD123+ basophils which degranulate following exposure to allergens or IgE receptor crosslinking agents.

Benefits and Applications

  • Screen compounds in vitro for their effect on degranulation by using the high throughput BAT assay
  • Measure potency of inhibitors on the degranulation of basophils by using BAT as a pharmacodynamic assay

Immune response monitoring using BAT pharmacodynamics assay to measure inhibitor potency
The figure shows the level of CD63 expression on basophils at baseline
and at various time points following drug treatment.

© 2017 Caprion