Immunotoxicity

and the Immune System

 

HemoGenix® is a Compliant Contract Research Service Supplier through Scientist.com

 

 

 Download the Immunotoxicity Contract Services Flyer

 

HemoGenix Contract Research Services

 

 

For all immunotoxicity testing services please contact HemoGenix® at contractresearch@hemogenix.com, or call (719) 264-6250. 

 

 

The majority of studies performed by HemoGenix® incorporate a Complete Service, Full Report that can be used for an IND application. More recently, Sponsors have requested a more streamlined study and studies involving high-throughput screening. For this reason, HemoGenix® now provides 3 types of study format. 

 

  1. Complete Service, Full Report Study: Fully customized study that includes the Study Plan, Draft Text and Final Text Report with QA audit.
  2. Rapid Toxicity Study: A customized study that includes the Study Plan and full protocol, raw results and graphical data in a single Excel Workbook. No formal text report and no QA audit is performed. This type of study is designed for early drug development. No interpretation or conclusions are provided.
  3. High-Throughput Screening Module (presently only available for human studies): A screening study is designed for high-throughput screening of compounds during ADME/Tox screening in multiples of 5 on specific cell populations to provide the most important ranking information. The full protocol, raw results and graphical data are provided in an Excel Workbook. No interpretation or conclusions are provided. This type of Screening Study is part of the ComparaTOX™ HT Platform.

HemoGenix® has developed several proprietary assays for in vitro immunotoxicity high-throughput screening and testing as well as other immune assays involving T- and B-lymphocytes and subsets. In addition, it also employs assays that have been developed by other companies and are commercially available. This list of assays and cell types are shown here and described in more detail below.

 

  • Lympho-hematopoietic stem cells and lymphopoietic progenitor cells
  • Lymphocyte proliferation/cytotoxicity assays 
  • Mixed lymphocyte culture/reaction (MLC/MLR)
  • Cytokine Release Syndrome (CRS) and Cytokine Release Assays (CRA)
  • Regulatory T-cell (Tregs) response
  • Cytotoxic T-cell (CTL) response
  • Antibody-dependent cell-mediated cytotoxicity (ADCC)
  • Antibody-drug conjugates (ADC)
  • Immunophenotyping
  • Macrophage (M1/M2) response
  • Phagocytosis, oxidative burst (neutrophils), and migration
  • NK activity 
  • Dendritic cell maturation and co-stimulation
  • Cell injury and cell death
  • Extracellular markers of activation

 

  • Human
  • Primate
  • Dog
  • Rat
  • Mouse

PLEASE NOTE that not all species may be available for particular studies.

Different target cells are used for different types of testing and assays.

  • Bone marrow (BM) is primarily used for studying responses to lympho-hematopoietic stem cells and lymphopoietic progenitor cells using our proprietary HALO®-Tox HT Platform as well as studies that involve B-lymphocytes.
  • Peripheral blood mononuclear cells (PBMNC) are used for many of the aforementioned testing, including MLC/MLR, immuno-oncology testing, immuno-phenotyping, cytokine release assays (CRAs) and many more.
  • Purified lymphocyte cell populations, both T-cells (e.g. CD3+, CD4+, CD8+) and B-cells, might be required for testing cytotoxicity, MLR and responses by T-regs, CTL and antibody responses.
  • Macrophages, dendritic cells (DC) and natural killer (NK) cells might be required fo co-cultures.
  • Primary cancer cells from CML, AML, MM, NHL and other cancers are now also available for immuno-oncology testing.
  • Lymphoid cell lines can also be incorporated into our testing procedures.

Small molecues can enter quiescent lympho-hematopoietic stem cells and can cause damage when these stem cells are called upon to proliferate. Small molecules can also affect primitive lymphopoietic progenitor cells. Large molecules, e.g. antibodies alone or conjugated with drugs can also be tested on these primitive cell populations.

 

The consequences of potential toxicity to lympho-hematopietic stem cells can be extreme since both lymphopoiesis and hematopoiesis can be damaged. The effect on lymphopoietic progenitor cells can affect the functionaling of the whole immune system.  

 

The following hematotoxicity assays are used to ascertain damage to these primitive and rare cell populations. For more information, please refer to the Hematotoxicity Page on this website.

  • HALO®-Tox HT for SC-HPP (Stem Cell - High Proliferative Potential), primitive lympho-hematopoietic stem cell populations. These stem cells give rise to both the lymphopoietic and hematopoietic system. Damage to these quiescent cells is predictive of damage to both systems. HALO®-Tox HT can be performed in both 96-well and 384-well plate formats. HALO®-Tox HT was the first standardized and validated high-throughput, hematotoxicity platform to be developed based on Suspension Expansion Culture™ (SEC™) and ATP bioluminescence technologies. It is the basis of all HemoGenix® proprietary ATP bioluminescence toxicity platforms.
  • HALO®-Tox TCP are specific progenitor T-cell (P-Tcell) toxicity assays. P-Tcell assays are performed on bone marrow mononuclear cells (MNC) and usually require the stimulation by IL-2 and co-stimulators, such as CD3 and CD28. HALO®-Tox TCP is part of the HALO®-Tox HT "Global" 7-Population Assay Platform.
  • HALO®-Tox BCP are specific progenitor B-cell (P-Bcell) assays in which IL-4 or IL-7 may be used individually or combined with other factors into a cocktail. For some species, P-Bcells can be determined in the unpurified MNC fraction of bone marrow. However, for human P-Bcells, the MNC fraction must be further purified in order to reduce the effects of other cell types on P-Bcell function. HALO®-Tox BCP is part of the HALO®-Tox HT "Global" 7-Population Platform.
  • HALO® Real Time is a non-lytic, bioluminescence real time assay used to determine the onset of toxicity to primitive stem and progenitor cells in a dose-dependent manner. It is also used to follow the growth kinetics of different cell types. HALO® Real Time can be simultaneously multiplexed with flow cytometric phenotyic analysis to determine which cell populations are affected.
  • ComparaTox™. A multi-tissue, multi-species comparative toxicity platform that incorporates both HALO®-Tox HT and ImmunoGlo™-Tox HT.
  • HALO®. For studies that do not involve toxicity screening or testing, but do rely on the detection of primitive lympho-hematopoietic stem cells and primitive T- and B-progenitor cells, the HALO® Assay is used.

Whereas HALO® is used for stem cells and primitive progenitor cells of the lymphopoietic system, ImmunoGlo™ and ImmunoGlo™-Tox HT assays focus on immune T- and B-cell proliferation studies and potential immunotoxicity to T- and B-cell populations.  

 

Most studies on T- and B-cells use peripheral blood mononuclear cells (PBMNC) as a target. If neither T- nor B-cells are are required, then the whole MNC fraction is used for measuring proliferation or cytotoxicity employing the following ATP bioluminescence assays with high-throughput capability.

  • ImmunoGlo™ would be used to determine a proliferative response.
  • ImmunoGlo™-Tox HT would be used to determine a cytotoxic response. 

 

For studies that specifically require the detection of T- or B-cell response, HemoGenix® has developed individual assays.

  • ImmunoGlo™ TCP or ImmunoGlo™-Tox TCP are specific Tcell proliferation/T-cell immunotoxicity assays. Depending on the species, individual cytokines or cytokines combined with co-stimulators, such as CD3 and CD28 will be used. In addition, if required, purified T-cell populations can be used as target cells. ImmunoGlo™-Tox TCP is part of the ComparaTox™ multi-tissue, multi-species, comparative toxicity platform that is available for high-throughput immunotoxicity screening. 
  • ImmunoGlo™ BCP or ImmunoGlo™-Tox BCP are specific Bcell proliferation/ B-cell immunotoxicity assays. Depending on the species, IL-4 or IL-7 is used and in some cases combined into a cocktail with other factors to stimulate B-cells. All human studies are now performed using a purified population B-cells to prevent the involvement of other cell types that might produce results that would be difficult to interpret. ImmunoGlo™-Tox BCP is also part of the ComparaTox™ Platform.
  • ImmunoGlo™ MLC is a 1- or 2-way mixed lymphocyte reaction (MLR) or culture (MLC) in which the stimulator cells are treated with mitomycin-C (as opposed to radiation) to inhibit their proliferation in a 1-way MLC. Like other ImmunoGlo™ assays, these Mixed Lymphocte Reactions incorporate an ATP bioluminescence readout, rather than a radioisotope, fluorescence or absorbance readout. Co-culture studies (described below) can also be considered mixed lymphocyte reactions.
  • ImmunoGlo™ Real Time is a unique assay that can be use to determine time-dependence, growth kinetics or the onset of  immunotoxicity in a dose-dependent manner and can be simultaneously multiplexed with flow cytometric phenotyic analysis to determine which cell populations are affected. ImmunoGlo™ Real Time is often used to follow the growth of specific cells types when expanded in culture.

 

All ImmunoGlo™ assay are available as 96- or 384-well plate studies. 

Cytokine Release Assays (CRAs) represent an important aspect of understanding whether a new drug candidate will elicit an inflammatory response. Performing CRAs on potential COVID-19 therapies has become more important to ensure that no potential complications occur as a result of patient administration.

When an inflammatory response is instigated by disease complications, or an adverse effects caused by a biological therapy that results in the massive induction and release of different pro-inflammatory cytokines that can be fatal to the patient. This occured in 2006 when a group of patients received TGN1412, a monoclonal antibody therapy directed against the CD28 receptor that was supposed to treat autoimmune disease and hematopoietic malignancies. Other regulatory approved therapeutics have also been shown to induce Cytokine Release Syndrom (CRS), otherwise known as The Cytokine Storm. These include muromanab, trastuzumab, blinatumomab, alemtuzumab and even some CAR-T therapies. 

 

Cytokine Release Assays are performed in vitro and are required by regulatory agencies to try and ensure safety of the new therapy, particularly when the therapy targets membrane receptors. 

 

There are many different variations of in vitro Cytokine Release Assays. HemoGenix has focussed and offers three main types of assay, depending on the type of molecule being tested.

  • Human Whole Blood CRAs: These recreate the in vivo situation for immune subsets and the distribution and expression of Fc gamma receptors. The test compound is added at different doses and compared to the effect of known positive and negative controls.
  • Human High Density Peripheral Blood Mononuclear Cell (PBMNC) CRAs: Whole peripheral blood is fractionated by density gradient centrifugation to produce the mononuclear cell (MNC) fraction, which is then is used at high density (1 x 107 cells/mL) in a 96-well plate assay. Like Whole Blood CRAs, the test compound is added in serial dilutions and compared to the effect of known positive and negative controls. This type of assay is also used to determine if a test compound can mitigate the cytokine storm by adding the compound together with known compounds that induce the cytokine storm.
  • Co-Cultures of PBMNC and Human Umbilical Vein Endothelial Cells (HUVEC) CRAs: These co-cultures are particular interesting because they not only allow the compound to be physiologically presented, but also take into account target cross-linking and FC-binding. In these CRAs, HUVEC cells are allowed to provide a confluent layer on the growth surface of 96-well plates. This is followed by the addition of PBMNCs and the addition of the serially diluted test compound and positive and negative controls.

 

In each of the above cases, the supernatants are collected. The presence and concentration of multiple cytokines is then determined using multiplex technology. The basic types of cytokines detected are IL-2, IL-6, IL-10, IFN gamma and TNF alpha. However, other cytokines can also be added including, IL-4, IL-8, IL-10 and many others.

What are Regulatory T-Cells (Tregs)?

Regulatory T-cells or Tregs are a specific CD4+ T-cell population that are involved in suppressing the immune response in autoimmune disease, transplantation and graft versus host (GvH) disease. The cells act through cell interaction and the secretion of cytokines (see also Cytokine Release Assays). Tregs express the CD25 or IL-2RA antigen as well as the transcription factor Foxp3. 

 

How are Tregs Determined and Used in Studies?

  • Tregs are usually detected by flow cytometry and their response is measured using ImmunoGlo™. 
  • A cocktail of CD4, CD24, Foxp3 and CD127 are used to detect regulatory T cells.
  • If Tregs need to be expanded for other assays, e.g. suppression by CTLs, this is performed CD3 and CD28 microbead Treg Expansion from Miltenyi Biotec in the presence of IL-2. Cell proliferation is detected using ImmunoGlo™.
  • Treg suppression is usually performed using a co-culture of Tregs and cells that might suppress Tregs, for example, CTLs.

 

What are Cytotoxic T Lymphocytes (CTL) and how are CTLs Determined and Used in Studies?  

Cytotoxic T-cells are effector cells important in the adaptive immune response. They respond to viral and bacterial pathogens, tumor cells and transplanted cells. There presence usually correlates with protective immunity. Three major factors are required for a CTL response, these being (1) antigen presenting cells (dendritic cells, see below), (2) CD4+ T helper cells and, (3) CD8+ T effector cells. Needless to say, the reaction is complex, especially when the antigen is unknown.   

 

Neverlethess, CTLs can be detected in several ways.

  1. Lymphocyte proliferation assay, designed to measure specific cells.
  2. Detection of CD4+, CD8+ and dendritic cells by flow cytometry.
  3. Production and secretion of specific cytokines due to stimulation.
  4. Intracellular cytokine detection in specific cells by flow cytometry.

 

Antibody-dependent cell-mediated cytotoxicity or ADCC allows the immune system to kill diseased cells such as cancer cells or viral-infected cells. The use of monoclonal antibody therapy utilizes the mechanism of ADCC in which the effector cell binds a specific monoclonal antibody (usually an IgG class) which, in turn, binds to the target cells forming a bridge that binds the target cell and triggers a lytic process that kills the target cell.

 

How ADCC is Measured 

  • HemoGenix® uses the Promega, ADCC Reporter Bioassay which uses a FcyRIIa-H, -F or -V variant receptor in an effector cell line with a luciferase reporter. Target cells are WILs-S or Raji cells. 
  • The engineered effector cells replace peripheral blood mononuclear cells (PBMNC) or natural killer (NK) cells, which can be highly variable.
  • Antibodies are the test articles and provided by the Sponsor.
  • The antibody is prepared in a serial dilution and added to the taget cells in a 96-well plate. A reference antibody might also be used. 
  • The engineered effector cells are added and cultures incubated for 6-24hr.
  • The response (if any), is determined using luciferase and the readout of a bioluminescence signal.
  • The presence of an ADCC mechanism is quantified using a 4-parameter logistic curve fit of the antibody dose response curve.

 

Antibody-drug conjugates are antibodies that target a specific cell types, usually a cancer cell, and are conjugated to a drug that kills the cancer cell. 

 

There are several questions that arise using ADCs. They include:

  1. Does the ADC also target normal cells besides the "targeted cells"?
  2. Is the "linker" that conjugates the antibody to the drug stable or does it break after administration releasing the drug and the antibody alone?
  3. Does the drug kill normal cells even when it is conjugated to the antibody?.

These are called "off-target" toxicities. 

 

All of these questions and more can be answered using HALO®-Tox HT or any of the ImmunoGlo™-Tox HT Platforms. In addition, other tissue toxicity assays may also come into play depending on the cells targeted and/or the cytotoxic drug used.

 

Depending on the microenviromnet in which a macrophage finds itself, it can respond to different functional programs. Indeed, macrophages can respond to numerous types of internal and external signals, from changes in partial oxygen tension to innate immune effector cells. This phenomenon is called "macrophage polarization". 

 

Macrophages are involved in numerous biological systems and responses. Macrophages are originally hematopoietic cells produced in the bone marrow and are intimately involved in the bone marrow microenvironment of stem cell regulation. However, during their development, they will enter different organs and tissues and become part of that organ or tissue. They are then known by a different name. For example, Kupfer cells or liver macrophages.

 

There are 2 groups of macrophages. 

  • M1 macrophgaes respond to classic activation by, for example, lipopolysaccharide (LPS), granulocyte-macrophage colony-stimulating factor (GM-CSF), tumor necrosis factor (TNF). They express NF-kB and AP-1 and secrete many different pro-inflammatory cytokines, such as, IFN-y, IL-1, IL-6, IL-12 and TNFa. They also produce reactive oxygen species ROS). They are also responsible for Cytokine Response Syndrome, otherwise called a "cytokine storm".  
  • M2 macrophages can be derived from M1 macrophages in response to excessive injury, whereby M2 macrophages respond to "alternative activation". This type of activation leads to an anti-inflammatory response with the production of IL-10, an immunosuppressive cytokine. 

 

The role of macrophages within a specific immune response can be investigated using multiple methodologies. The response of macrophages to different stimuli can be analyzed both by flow cytometry and the use of multiplexing assays such as LUMINEX. 

    

For specific studies on or using macrophages, please contact HemoGenix®.

Dendritic cells, also called professional antigen presenting cells or accessory cells, are of two main types namely, those derived from lymphocytes, called plasmacytoid DCs and those derived from monocytes, called conventional DCs or monocyte-derived DCs. 

 

Dendritic cells are idenfied by cell surface markers and the realease of cytokines. A small number of DCs are present in peripheral blood. However, to obtain DCs for studies, CD14+ monocytes are purified from peripheral blood mononuclear cells (PBMNC) by magnetic bead separation and expanded in GM-CSF and IL-4 to obtain immature DC with a phenotype of CD11+/CD14-. Kinetics of DC expansion is followed using ImmunoGlo™ Real Time Assays. Dendritic cell maturation continues in culture and is followed by phenotypic expression of DC-specific surface antigens. Function is determined by the release of cytokines.

 

Dendritic cells can be used in mixed lymphocyte reactions using ImmunoGlo™-MLC and many of the assays described above. They can also be co-cultured with NK cells (see below).

    

For specific studies on or using DCs, please contact HemoGenix®.

Natural Killer cells or NK cells play a major role in innate immunity and are also found in the peripheral blood. They are highly cytotoxic. They are present in lymphoid organs, e.g. lymph gland and tonsils and are avid cytokine producers. These cells have a phenotype of CD56+ and can be CD16+ or CD16-. This does not mean to say that other membrane markers are not available. Besides numerous cytokines, NK cells also secrete granzyme and perforin.

 

Expansion of NK cells for studies can take several weeks and is performed either by co-culture with mitomycin-c - inhibited K562 cells or in the presence of IL-2. The presence of NK production is followed using phenotypic analysis and/or ImmunoGlo™ Real Time assays.

   

For specific studies on or using NK cells, please contact HemoGenix®.

The assays/readouts already described for immunotoxicity testing and analysis of the immune system are:

  • HALO® family of assays
  • ImmunoGlo™ family of assays
  • Luciferase bioluminescence
  • Immunophenotyping and intracellular cytokine detection by flow cytometry
  • LUMINEX. 

 

HemoGenix® has many other assay capabilities including, but not limited to:

  • Mitochondrial disfuntion
  • Oxidative DNA damage
  • Oxidative stress
  • Membrane leakage
  • Apoptosis
  • Growth kinetics
  • Cell quality and potency of cell therapy products
  • Potency of drugs
  • Other Mechanism of Action assays

 

For more information of HemoGenix testing capabilities, please contact HemoGenix®.

You can perform immunotoxicity assays in-house using assay kits that are sold by Preferred Cell Systems™. Please click on the links below to take you to the assay kit page on the Preferred Cell Systems website.

 

ImmunoGlo™: ATP bioluminescence immune cell proliferation assays.

ImmunoFluor™: Fluorescence immune cell proliferation assays.

ImmunoLight™: Absorbance immune cell proliferation assays.

ImmunoGlo™-Tox HT: General immunototoxicity assays.

ImmunoGlo™-ToxTCP: T-cell cytotoxicity assays.

ImmunoGlo™-Tox BCP: B-cell cytotoxicity assays.

ImmunoGlo™ MLC: ATP bioluminescence 1- or 2-way mixed lymphocyte culture assays.

ImmunoFluor™ MLC: Fluorescence 1- or 2-way mixed lymphocyte culture assays.

ImmunoLight™ MLC: Absorbance 1- or 2-way mixed lymphocyte culture assays.

HALO®-Tox HT: Hematotoxicity assays.

HALO®: ATP bioluminescence lympho-hematopoietic stem and progenitor cell assays.

HemoFLUOR™: Fluorescence lympho-hematopoietic stem and progenitor cell assays.

HemoLIGHT™: Absorbance lympho-hematopoietic stem and progenitor cell assays.