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Dan L. Duncan Cancer Center

Houston, Texas

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Dan L. Duncan Cancer Center
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MHC Tetramer Shared Resource Order Guidelines

Why MHC Tetramers?

T cells play essential effectors and regulatory roles in adaptive immune responses to tumors, viruses, bacteria, parasites, transplanted tissues, allergens and even to self antigens. T cells use the T cell antigen-receptor (TCR) to recognize their antigens, which are often in the form of peptides bound to major histocompatibility complex molecules (MHC), also called HLA (human leukocyte antigens) in humans.

Desire to identify antigen-specific T cells through the MHC/peptide complex was always there. However, the inherent affinity between MHC and TCR was demonstrably weak and with short half-life, providing an explanation for the failure of many efforts to obtain staining of T cells with monomeric MHC. In the 1994-1995, this problem was solved by the development of a revolutionized technique called “MHC Tetramer”, which bind strongly to antigen-specific T cells due to an increased avidity effect.

Nowadays the MHC Tetramer technique has become a "gold standard" for the quantification of T cell immune responses. The acceptance of the method is indicated by the number of citations to key papers describing the method: the original Science paper describing the method has been cited more than 500 times.

By offering exquisite antigen specificity and sensitivity, it is suitable for basic and clinical study in a number of applications including cancer prevention, cancer therapy, cell and gene therapy, immunotherapy, drug / vaccine development, and non-cancer related immunology research. Proven through extensive research, MHC Tetramer technology has been used to accurately and efficiently monitor T cell responses in the following conditions:

  • Viruses (Human Immunodeficiency Virus-Acquired Immunodeficiency Syndrome, Epstein Barr Virus-mononucleosis, Cytomegalovirus, Human Papilloma Virus, Hepatitis B, Hepatitis C, Influenza, Measles and viruses for Rhesus Macaque and Murine)

  • Parasitic infection (malaria)

  • Cancer (breast, prostate, melanoma, colon, lung, cervical)

  • Autoimmune diseases (multiple sclerosis, rheumatoid arthritis)

  • Transplantation

The major benefits of using this method have been summarized as the following:

  • Rapid quantitative results

  • No radioisotopes or in vitro stimulation and expansion

  • Exquisite antigen specificity with high sensitivity

  • Maintenance of cell integrity for further analysis

Fluorescent Label

All reagents are labeled with either commercially manufactured streptavidin-allophycocyanin (SA-APC) or streptavidin-phycoerythrin (SA-PE) fluorophores. These labels were chosen for use based on experience from several laboratories using tetramers. Some lot-to-lot variation in SA-PE has been observed, but should not seriously affect tetramer sensitivity or background if the staining suggestions (provided with the product) are followed. If other labeling reagents are desired, they must be provided by the requestor.

b2-microglobulin

We can provide MHC class I tetramers with either human or murine b2-microglobulin. However, please note that we typically fold all tetramers of every species with human b2-microglobulin. Use of murine b2-microglobulin may reduce the stability of the reagent. Typically staining of murine CD8+ T-cells with tetramer is NOT affected by the use of human b2-microglobulin.

Risk of Failed Productions

One of the factors that determine the success of the tetramer production is the binding affinity of the peptide for the MHC class I molecule. Unfortunately, we can not accurately predict the binding properties of a peptide according to its sequence. We also do not have specific numbers that would represent a binding threshold for the peptide. Therefore, the failure of tetramer production happens occasionally. In that case, a second attempt for the MHC/peptide folding and chromatographic purification will be tried with no additional charge. If the 2nd attempt also fails, we will terminate the preparation and the order will be partially charged.

Some online databases can help to predict the binding affinity between MHC molecules and peptides. Below are two examples. The positive result returned from searching these databases may not guarantee a successful tetramer production but can be used as a good reference.

SYFPEITHI Epitope Prediction

HLA-Peptide Binding Predictions

Quality Control

Quality control of tetramer reagents produced includes the following:

  • All tetramers will be purified by size exclusion and ion exchange chromatography. FPLC chromatograms of the reagent demonstrating proper folding and multimerization will be recorded.

  • Each tetramer is tested for the level of biotinylation.

  • ELISA using anti b2-microglobulin antibodies specific for properly folded MHC I.

Please direct ordering status inquiries or technical questions to Lily Wang at (713) 798-3918 or lxwang@bcm.edu.

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