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and the Immune System

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There are essentially two parts to investigating immunotoxicology and the immune system as a whole.

 

The first deals with the stem cells common to both the lymphopoietic and hematopoietic lineages, the definitive lymphopoietic stem cells and their progeny, the lymphopoietic progenitor cells, which, in turn, give rise to the different T- and B- lymphocyte populations.

 

The second part focusses on the the T- and B- lymphocyte lineages, lymphocyte subpopulations and the response of these subpopulations to perturbations, drugs and xenobiotics. 

 

 Step 1: Primitive Lympho-Hematopoietic Stem Cells and Immunotoxicity

A stem cell common to both the lymphopoietic and hematopoietic lineages can provide predictable information as to whether a compound will affect the cells of the lymphopoietic lineages (T and B). This stem cell population is referred to as the High Proliferative Potential or HPP stem cell. Its position in the organizational hierarchy of the blood-forming system is at the point where lymphopoiesis and the immune system diverse from the hematopoietic system. In other words, this stem cell is extremely primitive.

 

In fact, the stem cell HPP (SC-HPP) is usually in the quiescent state. Even though this stem cell population is quiescent, small molecules can still enter these cells resulting in serious probelms. If the small molecules affect the proliferation process, when called upon to re-enter the cell cycle and proliferate, these small molecules could become cytotoxic and kill the primitive stem cells. The consequence would be that the lymphopoietic and immune system (not to mention the hematopoietic system) may not be able to repopulated. This, in turn, would cause serious downstream biological and clinical failures.

 

Assays to detect the effect on primitive stem and progenitor cell populations usually inorporate the HALO® Platform, usually HALO®-Tox HT or HALO®-Real Time, both of which can be multiplexed with many other assay endpoints.

  

 Step 2: Immunotoxicity Testing

The second step of in vitro immunotoxicity testing, or to investigate specific functional characteristics of the immune system, involves a number of assays for developing or mature T- and B-lymphocytes. These include, but are not limited to:

  • Lymphocyte proliferation/cytotoxicity assays 
  • Mixed lymphocyte culture/reaction (MLC/MLR)
  • Regulatory T-cell (Tregs) response
  • Cytotoxic T-cell (CTL) response
  • Antibody-dependent cell-mediated cytotoxicity (ADCC)
  • Antibody-drug congugates (ADC)
  • Immunophenotyping
  • Macrophage (M1/M2) response
  • Phagocytosis, oxidative burst (neutrophils), and migration
  • NK activity 
  • Dendritic cell maturation and co-stimulation
  • Cytokine/chemokine production and release
  • Cell injury and cell death
  • Extracellular markers of activation

 

The assays above can be performed using:

  • Peripheral blood mononuclear cells (PBMC)
  • Bone marrow (BM)
  • Purified lymphocyte cell populations

 

Species include:

  • Human
  • Mouse
  • Please enquire for other species

 

Many of the above tests incorporate ImmunoGlo™-96, ImmunoGlo™-Tox HT or ImmunoGlo™-Real Tme, which can be multiplexed with other assay endpoints.

 ImmunoGlo™-Tox HT, ImmunoGlo™-MLC and ImmunoGlo™-Real Time

 

  • ImmunoGlo™-Tox HT is a lytic, ATP bioluminescence readout used an end-point readout.
  • ImmunoGlo™-Tox RT is a non-lytic, bioluminescence real time readout to determine the onset of immunotoxicity in a dose-dependent manner and can be simultaeously multiplexed with flow cytometric phenotyic analysis to determine which cell populations are affected by the insult.
  • ImmunoGlo™-MLC was designed for mixed lymphocyte cultures or reactions in which the stimulator cells are treated with mitomycin-C (as opposed to radiation) to inhibit their proliferation, but not they ability to interact with other cells. 
  • Both ImmunoGlo™-Tox RT and ImmunoGlo™-Tox HT can be combined with each other to produce highly accurate determinations of potential toxicity and associated measurement parameters.
  • Both assays have high-throughput (96- or 384-well plate format) screening capability of potential immunotoxicants for ADME-Tox or later stages of drug development, to help reduce unexpected immune responses during pre-clinical testing and clinical trials.
  • Both assays are used to determine the effects of xenobiotic agents on immune cells.
  • Predictive in vitro assay platforms to determine the response of lymphopoietic stem and progenitor cells to drugs and other xenobiotic agents that would indicate potential cytotoxicity in downstream immune cell populations.
  • Drug-drug interations (DDI) on specific lymphopoietic cell populations.
  • A 3Rs Alternative Assay Platform for Reduction, Refinement and Replacement of animal testing.
  • Comparison and ranking of immune responses in multiple species.
  • ImmunoGlo™-Tox HT incorporates the most sensitive ATP bioluminescence readout available to measure proliferation, cytotoxicity, cell number and even apoptosis. (10-100 times more sensitive than WST-1, CSFE and other absorbance or fluorescence readouts).
  • ImmunoGlo™-Tox HT is a calibrated and fully standardized assay that allows results to be reliably compared over time.
  • Rapid turnaround time: ImmunoGlo™-Tox RT results can usually be obtained in 2-4 days. ImmunoGlo™-Tox HT results are usually obtained in 4 to 7 days depending on species.
  • Both ImmunoGlo™-Tox HT and ImmunoGlo™-Real Time assays have been designed for multiplexing with other assay readouts using the same sample.

 Immunotoxicity Testing Panel


Immunotoxicity Testing Panel 

Assay Name Assay Type Pathway Readout
HALO®-Tox HT Intracellular ATP (iATP) Stem cell, T- and B-Progenitor Cell Proliferation (Step 1) Bioluminescence
HALO®-Tox Real Time (RT) Reduction potential Stem cell, T- and B-Progenitor Cell Proliferation (Step 1) Bioluminescence
HALO®-Tox DDI Intracellular ATP (iATP) Stem cell, T- and B-Progenitor Cell Proliferation (Step 1 or 2) Bioluminescence
CAMEO™-96 Clonal (colony formation) & iATP  Stem cell, T- and B-Progenitor Cell Proliferation and Differentiation (Step 1) Colony number / Bioluminescence
CAMEO™-4 Clonal (colony formation) Stem cell, T- and B-Progenitor Cell Differentiation (Step 1) Colony number (manual)
ImmunoGlo™-Tox HT iATP

T- and B-Cell Proliferation

(Tier 2)

Bioluminescence
ImmunoGlo™-Tox Real Time (RT) Reduction potential

T- and B-Cell Proliferation

(Tier 2)

Bioluminescence
ImmunoGlo™-MLC iATP

T- and B-Cell Proliferation

(Tier 2)

Bioluminescence
Cell cycle DNA marker Proliferation (Tier 2) Fluorescence
FlowDiff™ Immune membrane expression markers (e.g. CD3, 4, 8, 19, 73) Characterization / Differentiation (Tier 2) Fluorescence
LIVEGlo™ iATP Metabolic viability / Mitochondrial Function (Tier 1 & 2) Bioluminescence
Dye Exclusion Viability 7-Aminoactinomycin D (7-AAD) / Propidium Iodide (PI) Membrane integrity (Tier 1 & 2) Fluorescence
Mitochondrial ToxGlo™* ATP Mitochondrial dysfunction (Tier 2) Bioluminescence / Fluorescence
Glutathione Assay Glutathione Oxidative stress (Tier 2) Luminescence
OxyFLOW™ 8-Oxoguanine addudcts Oxidative DNA damage (Tier 2) Fluorescence
CaspaseGlo™* Apoptosis Caspases (Tier 2) Luminescence
Annexin-V / PI Apoptosis / Necrosis Phosphatidylcholine (Tier2) Fluorescence
GFkine™  Growth factor, cytokine production / release Multiple (Tier 2) Multiple

* Promega Corporation assays 

 

If an assay is not listed, please contact HemoGenix® to see if it might be available or can be developed and validated for a specific study.