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MuLV gp70 (AH1) Tetramer - a great tool for observing antigen specific immune response in cancer

Posted by M. Mamunur Rahman, PhD on Jun 25, 2020 12:00:00 PM

MuLV gp 70, also designated as AH1 (SPSYVYHQF), is an endogenous viral envelope glycoprotein encoded in the mouse genome that is transcriptionally silent in normal tissues. The expression of gp70 is reactivated in a few cancer cell lines including CT26 colorectal cancer and B16 melanoma cells. Other investigators have demonstrated the presence of MuLV-encoded molecules in murine lymphomas and leukemias as well as Simian Virus 40 (SV40)-transformed NIH-3T3 cells. Researchers measure total T cell and T cell subset by detection of various cell surface molecules to gain better insight into the central role of the immune system. MBL International offers MHC tetramer reagents for murine model antigens, tumor antigens, viral antigens, etc. MHC-peptide tetramer complexes detect antigen specific T-cells at the single cell level, allowing for accurate measurements of the functional immune response in cancer, infectious diseases, and automimmune diseases.

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Topics: Tetramer

Intracellular Staining with MHC Tetramers

Posted by Dennie Magcase on May 27, 2020 1:00:00 PM

T-cells recognize peptides/MHC complexes (pMHC) through the T-cell receptor (TCR). This is the first step for the initiation and shaping of protective immunity against viruses and tumor antigens. Fluorescently labelled pMHC tetramers have dramatically transformed the detection of antigen specific T-cells. MHC tetramer staining of antigen specific T cell clonotypes is detected by flow cytometry to probe T-cell responses1 and to further characterize antigen-specific T cells, for example, to study the surface markers as well as intracellular proteins. The measurement and analysis of effector function of antigen-reactivated T cells is now possible by MHC tetramer and flow cytometry-based intracellular cytokine staining (ICS). This method allows concurrent phenotypic characterization and cytokine detection within single cells2.

The production of cytokines plays an important role in immune response.  Cytokines are involved in many pathways, including the induction of many anti-viral proteins by IFNγ, the induction of T cell proliferation by IL-2, and the inhibition of viral gene expression and replication by TNFα3.  Cytokines are not preformed factors but are produced and secreted in response to cellular activation.

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Topics: Tetramer, cytokine

In vivo NIRF whole-body imaging through MHC tetramers

Posted by Yuri Poluektov on Apr 8, 2020 1:00:00 PM

The use of MHC tetramers for the detection of antigen specific T cells has been a well-established technique since it originally gained prominence in the mid-1990s. Since then, the sophistication of MHC tetramer and multimer design as well as its sensitivity in detecting target T cells has been steadily improving. Most applications of MHC Tetramers involve the use of Flow Cytometry to enumerate, purify or sort specific T cells, but in some cases, it is possible to use tetramers to visualize entire immunological processes taking place inside a whole body.

We want to bring to your attention a recent paper by Welsh and Song et. al. (Plos Biology Feb, 2020)1. In their study, while constantly using MHC Class II tetramers to detect and enumerate pathogenic T cells, responsible for the progression of Rheumatoid Arthritis (RA) as well as Experimental Autoimmune Encephalomyelitis (EAE) induced in mice models, the group performed a number of imaging trials. This imaging procedure involved a modified in vivo NIRF whole-body imaging technique described in the paper. The group was successful in identifying a co-localized population of Collagen peptide specific T-cells and a molecular probe specific for denatured Collagen protein molecules using an IRDye800CW-conjugated CII(280–294) peptide loaded HLA-DR1 tetramer.

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Topics: Tetramer, QuickSwitch

Screening for COVID-19 T-cell peptides and immune monitoring with MHC tetramers in a single assay

Posted by Pirouz Daftarian, Ph.D., Marc Delcommenne, Ph.D. on Mar 17, 2020 3:00:00 AM

COVID-19 infected cells can be recognized by T-cells only after SARS-CoV-2 peptides are processed and presented in the context of self MHCs.

Identifying these peptides have essential utilities:

  • Immune monitoring: Using peptide-MHC tetramers to assess vaccine-induced immunity
  • Designing potent vaccines that elicit durable responses
T-cells are activated only by interacting with processed peptides (e.g. of SARS-CoV-2) that sit in the groove of MHC Class I and II molecules. Identifying these peptides can be used (in forms of peptide-MHC tetramers) to assess vaccine induced immunity and assist in designing potent vaccines with durable responses.
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Topics: Tetramer, Peptide, covid-19, SARS-CoV-2, vaccine, MHC

Cancer Immunotherapy - Growing focus on CD4+ T lymphocytes and MHC Class II Neoantigens

Posted by Anthony G Pietrantoni on Feb 11, 2020 1:00:00 PM

It is now well accepted that CD8+ T cells play a central role in mediating anti-tumor immunity by recognizing tumor-associated antigens presented on major histocompatibility complex class I by their expressed T cell receptor.  Majority of studies on cancer neoantigens have therefore focused on MHC class I restricted antigens recognized by CD8+ T cells.   However it is also becoming increasingly clear that CD8+ T cell binding neoantigens are not sufficient for mounting a powerful anti tumor response. The recent paper by Alspach et al. proved that spontaneous and immunotherapy-induced antitumor responses required presence of CD4+ in addition to CD8+ T cells (1).

Multiple studies indicate that CD4+ T cells mediate antitumor effects through mechanisms that vary according to tumor environments whether they act as helper or cytotoxic cells but are ultimately dependent on MHC class II neoantigens.   In some instances CD4+ T cells were shown to acquire cytotoxicity activity in vivo in presence of a high level of IL-2 or when IL-2 sequestering T Reg cells were absent (2).  A recent investigation reported that cytotoxic CD4+ T cells are particularly abundant among supercentenarians and reached 25% of T cells on average, suggesting they are essential to protect against infections and cancers, contributing to achieve an extended disease-free life expectancy (3).  Therefore, with improved understanding of the intricate interactions between cytotoxic and helper CD4+ lymphocytes with tumors and antigen presenting cells, interest is currently focused on MHC class II dependent immune activation mechanisms.  MHC class II restricted neoantigens have now the potential to be promising targets of cancer immunotherapies.

Learn more about Class II Tetramers

 

References

(1)Alspach, E. et al. MHC-II neoantigens shape tumour immunity and response to immunotherapy. Nature 574: 696–701 (2019).

(2) Śledzińska, A. et al. Regulatory T Cells Restrain Interleukin-2- and Blimp-1-Dependent Acquisition of Cytotoxic Function by CD4+ T Cells. Immunity. 52(1):151-166 (2020).

(3) Hashimoto K. et al. Single-cell transcriptomics reveals expansion of cytotoxic CD4 T cells in supercentenarians. Proc Natl Acad Sci USA. 116(48):24242-24251 (2019).

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Topics: Tetramer, Cancer

Is your drug candidate able to reverse the T Cell Exhaustion-like state in our in vitro functional screening assay?

Posted by Pirouz Daftarian, Ph.D. on Dec 12, 2019 1:00:00 PM

The goal of cancer immune checkpoint therapies is to cure tumor-specific T cells from dysfunction which is caused by elements from tumor deposits.  In this blog, we briefly describe the process of T cell exhaustion and how it can be harnessed for immuno-oncology drug discovery and more specifically to screen immune checkpoint drug candidates. First, the current function-based characterization methods for immunomodulatory drug candidates will be summarized. We have selected three methods which mimic prerequisite parameters of early T cell exhaustion (TEX). Next, we briefly describe an improved recall antigen-based potency assay as detailed elsewhere. Lastly, we describe an in vitro reversible early TEX-like  model, which may be tailored with cancer-induced T cell suppression agents.  Here it should be noted that cancer induced T cell dysfunction is also referred to as TEX, covering a broad spectrum of different molecular T cell pathologies. The term “T cell exhaustion” in this review mainly refers to an early stage of this process.  We have devised an in vitro model that recapitulates components of tumor deposits responsible for inducing TEX.

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Topics: Tetramer, Cancer, t cell

New tetramer for vaccine research targeting Tuberculosis and Ag85B specific T-cells

Posted by Bindi M. Doshi, PhD on Jul 23, 2019 1:00:00 PM

Tuberculosis (TB) is caused by the Mycobacterium tuberculosis and usually targets the lungs.  This disease is spread when an infected person sneezes or coughs, thereby releasing small droplets into the air and passing to a new individual. When left untreated, death can occur. Thankfully, there is a vaccine for TB called Bacille Calmette-Gurin (BCG) and is often given to young children.  This vaccine has been shown to last 15 years. There is some doubt about the efficacy of the BCG vaccine in adult pulmonary TB1,2.  For this reason, more research needs to be done to provide a safe and effective TB vaccine for adults.

Antigen (Ag) 85 is gaining interest among vaccine development research.  Ag85 is preserved in Mycobacterium and allows bacteria to evade the host immune response by preventing mechanisms involved in terminating the infection3.  Research continues to uncover exactly how Mycobacterium tuberculosis persists and remains in an infected person while evading the immune system.  New advances in immunology are giving researchers a greater picture for how the immune response is manipulated by this bacteria.  It is clear that with greater understanding of how the human immune response system functions and how different infections manipulate this system, that targeted therapies, including vaccines, can be developed to combat the infection successfully.

MBL International is pleased to announce the launch of our new tetramer targeted to Ag85B-specific T cells for murine studies. As always, our team is on hand for any question or request that you may have.

Product Code Target Conjugate
TS-M719-1 I-Ab Mtb Ag85B240-254 Tetramer-FQDAYNAAGGHNAVF PE
TS-M719-2 I-Ab Mtb Ag85B240-254 Tetramer-FQDAYNAAGGHNAVF APC

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(1) Davenne,T., & McShane, H. (2016). Why don't we have an effective tuberculosis vaccine yet?. Expert review of vaccines15(8), 1009–1013. doi:10.1586/14760584.2016.1170599

(2) Andersen, P., & Doherty, T.M. (2005). The success and failure of BCG - implications for a novel tuberculosis vaccine. Nature Reviews Microbiology,3(8), 656-662. doi:10.1038/nrmicro1211

(3) Babaki, M. K., Soleimanpour, S., & Rezaee, S. A. (2017). Antigen 85 complex as a powerful Mycobacterium tuberculosis immunogene: Biology, immune-pathogenicity, applications in diagnosis, and vaccine design. Microbial Pathogenesis, 112, 20-29. doi:10.1016/j.micpath.2017.08.040

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Topics: Tetramer, Immunology, tuberculosis

QuickSwitch, a tool for adoptive T cell transfer

Posted by Yuri Poluektov on Apr 24, 2019 1:00:00 PM

Our understanding of how the immune system responds to cancer has increased by leaps and bounds in the past two decades, allowing us to develop a new approach to fight cancer that uses the power of the body’s own immune system to prevent, target, control, and eliminate the disease. Collectively, this technique of battling cancer is known as cancer immunotherapy. Such an approach encompasses many methods from broad ones like applying immune checkpoint blockers (ICB) or using monoclonal antibodies (mAbs) that target known cancer antigens to more personalized ones like adoptive T cell transfer including the use of Chimeric antigen receptor (CAR) T cells.

It is this personalized immunotherapy approach that has sparked a great deal of interest among researchers. This method involves the extraction of a patient’s T cells and the subsequent stimulation of the rare T cells populations that can fight cancer with specific antigens. For this form of therapy to work there needs to be an exact knowledge of the peptide sequences derived from tumor cells (neo-epitopes) that can be used to artificially stimulate a patient’s T cells to fight them.

Previously, we have described how by using the QuickSwitchTM Quant Platform peptides, they can be tested for their ability to bind MHC Class I molecules. This test does not involve prediction algorithms or large swaths of data with a great deal of false positives but provides an in vitro result of whether or not the peptide can be presented on the MHC molecule in about 8 hours. It is our hope that by using our kit, researchers will be able to discover and validate neoantigen peptide sequences more effectively, which would open up new ways for adoptive T cell transfer in cancer immunotherapy.

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Topics: Tetramer, QuickSwitch

Gating strategy for tetramer analysis to eliminate non-specific event

Posted by Wushouer Ouerkaxi, PhD, MD on Nov 14, 2018 1:00:00 PM

While MHC tetramer staining of antigen specific T cells for flow cytometry is considered highly specific, it could still be associated with non-specific staining. The gating strategy shown below is an example that investigators have used to reduce non-specific staining events using a dump channel. A dump channel can be planned on a case by case basis, on the experimental design, and consists of a pool of antibodies against cells that do not express our specific targets. For example, CD8+ T cells, anti-CD4/anti-CD14/anti-CD19 may be used to eliminate these immune cells from our targeted gate, and all may be labeled with the same fluorochrome. 

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Topics: Tetramer, Flow Cytometry

Which tetramers can be used for my mouse samples?

Posted by Tomohiro Nakajo on Aug 28, 2018 1:00:00 PM

The thymus shapes a host's T-cell repertoire so they do not respond to "self" antigens, yet are able to recognize "non-self" antigen challenges (from pathogens and tumors). T cell participation in these immune responses are dependent on interaction with peptide- and self-MHC. In other words, T cells only recognize the peptides derived from cancers or pathogens if they are embedded in the cleft of self host MHC grooves. Such peptides are processed and presented on the surface of antigen-presenting cells (MHC class I and II in dendritic cells, macrophages, and B cells) or all nucleated cells (MHC class I) allowing T cells to identify infected or cancer cells. Direct detection of such Ag-specific T cells and functional analysis is important for understanding disease immunopathogenesis and the process of drug development. MBL International has had tremendous success in advancing the methodologies to accurately measure antigen-specific T cells. 

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Topics: Tetramer

Quick!  Switch out your peptide! 

Posted by Marc Delcommenne, PhD on Mar 27, 2018 12:58:00 PM

The immunotherapy field is making great progress in aiding many disease treatments.  The hope is that one day, personalized immunotherapy can be used so each patient can use their own immune system to overcome their specific disease affliction.  QuickSwitch is a tool for creating custom MHC class I tetramers quickly and easily.

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Topics: Tetramer, QuickSwitch, Peptide, Peptide Exchange

Quick!  Switch out your peptide!

Posted by Marc Delcommenne, PhD on Jan 20, 2017 11:00:21 AM

The immunotherapy field is making great progress in aiding many disease treatments.  The hope is that one day, personalized immunotherapy can be used so each patient can use their own immune system to overcome their specific disease affliction.  QuickSwitch is a tool for creating custom MHC class I tetramers quickly and easily.

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Topics: Tetramer, QuickSwitch, Peptide, Peptide Exchange

Don't be fooled. Not all tetramers are created equal!

Posted by Bindi M. Doshi, PhD; Cheryl A. Guyre, PhD on Aug 24, 2016 11:45:03 AM

Have you seen comparisons of multimer technology claiming to be superior to tetramers? Be sure to ask which tetramers were used in the comparison! MBL tetramers have a clear advantage over academic tetramers and other commercial MHC multimer products, not only due to the reliability and high quality for every lot produced, but also due to the proprietary alpha-3 mutation. This mutation, engineered into the heavy chain of all of our human and macaque class I tetramers, helps decrease non-specific binding, leading to enhanced specificity. Check out the images below to see the details of this important technology!

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Topics: Tetramer

Help! My mouse tetramer stained ALL the CD8+ T cells!

Posted by Cheryl A. Guyre, Ph.D. on Aug 26, 2015 3:39:00 PM

You’re sitting at your flow cytometer, staring at your CD8 x tetramer plot in anticipation of a tiny but meaningful group of dots appearing in the upper right quadrant. You hold your breath, click “acquire,” and watch… Wait, what? What’s this ugly diagonal?? How come ALL the CD8 positives are dual positive??! Hey, this is my control mouse! That one should be completely tetramer negative! Yep, I’ve been there too. Classic case of using a CD8 antibody that doesn’t play nice with tetramers.

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Topics: Tetramer

Be positive! 5 ways to confirm your MHC tetramer is binding

Posted by Cheryl A. Guyre, Ph.D. on Apr 17, 2015 4:35:17 PM

In the previous Tetramer Tips blog, I suggested ways for you to be a negative Control Freak. Now it’s time to think positive! A positive control for a tetramer is a sample that contains cells expressing the specific T cell receptor of interest, i.e. has the exact specificity of the tetramer. Okay, brace yourselves; I’m going to be frank here.  Having a positive control for tetramer experiments is often a quest for the Holy Grail. In many (most, in fact) cases, you will not have access to a positive control, unless one of your experimental samples happens to show a positive result. In an experiment where no positive events are seen and no positive control was used, you cannot necessarily conclude that the donor/patient/mouse is negative for that T cell specificity, because, heck, maybe you got distracted while pipetting and forgot to add the tetramer to your staining cocktail!  A tetramer experiment with no positive control and no positives in the experimentals is, therefore, uninterpretable. This is the hard truth we must face.

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Topics: Tetramer, Immunology

Tetramer Tips for Success: How to be a (Negative) Control Freak

Posted by Cheryl A. Guyre, Ph.D. on Mar 20, 2015 10:48:00 AM

While being a Control Freak may not be the best choice for living in spiritual harmony, it is a downright asset in flow cytometry research!  Controls not only help you set up an experiment to get a clear or “true” answer, they can also help you troubleshoot what may have gone wrong when your data just doesn’t look quite right, so that your next attempt will turn out better.  In flow cytometry, controls are critical to help determine “real” events from artifacts.  So, what should you use as controls in tetramer experiments?

 

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Topics: Tetramer, Immunology

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