What is MHC Tetramer?

MHC Tetramer is a kind of immunological reagent. It consists of four major histocompatibility complexes (MHC) which bind to antigen peptides and are labeled with fluorescence for T cell immunoassay. MHC is composed of three kinds of molecules: I, II and III. It is related to immune recognition, rejection and immune response. It is a key molecule for antigen presentation. After recognizing antigenic peptide, MHC binds with antigenic peptide to form peptide-MHC complexes (pMHC) for recognition of corresponding T cell receptor (TCR) and activation of corresponding immune response. Therefore, it is a new trend to detect T cell epitopes by combining pMHC with TCR.

Basic Principles of MHC Tetramer Technology

In vivo, TCR on T cells can accurately identify the antigenic peptides binding to MCHI molecules on the surface of target cells. If we simulate the process in vivo, construct MHC-peptide complex in vitro and bind it specifically to TCR of CTL, we can directly detect antigen epitope-specific T cells. However, the affinity of TCR binding to specific MHC-peptide complex is very low, and the dissociation speed of TCR and MHC-peptide complex is very fast. The half-life of TCR binding to specific MHC-peptide complex is about several seconds to several minutes, which makes it difficult to detect antigen-specific CTL. Some researchers have realized that the dissociation rate of MHC-peptide complex with T cells can be greatly slowed down by constructing MHC-peptide complex polymer in vitro, so they have created MHC-tetramer technology based on biotin avidin cascade amplification principle.

Basic Process of MHC Tetramer Technology

The basic process of MHC Tetramer technology is: adding 15-amino-acid-length biotin substrate peptide (BSP) to the carboxyl end of HLA A2 antigen heavy chain to form fusion protein by genetic engineering technology; the fusion protein is co-incubated with beta-microglobulin and specific antigenic short peptide in a certain proportion in vitro to fold into the correct conformation and become MHC-peptide complex. Compounds: MHC peptide complexes were purified, and biotin was labeled on lysine residues of BSP, then biotin-labeled MHC-peptide complexes were combined with fluorescein-labeled streptavidin in 4:1, which formed the binding of MHC-tetramer and TCR on antigen-specific CTL. Finally, antigen-specific CTL was analyzed by flow cytometry.

Application of MHC-Tetramer

After the emergence of MHC-tetramer, it has been rapidly used in many fields. At present, the research on tumors, viral infections, autoimmune diseases and so on has achieved the most results. At present, the application of tetramers mainly focuses on the following aspects.

(1) Quantitative detection of antigen-specific CTL

MHC Tetramer can quantitatively detect antigen-specific CTL with high specificity and sensitivity. By understanding the quantity and variation of antigen-specific CTL and exploring the role of antigen-specific CTL in the pathogenesis, we can screen specific CTL immunodominant epitopes, lay the foundation for vaccine development, evaluate the immune effect of vaccine, and provide clear indications for clinical observation and treatment.

(2) Phenotypic analysis of MHC Tetramer positive cells

The expression of surface molecules on MHC-tetramer positive cells was analyzed by flow cytometry combined with multi-fluorescent labeling, or some effector molecules or related markers were detected by intracellular staining.

(3) Isolation of T cells

The antigen-specific T cells labeled with MHC-tetramer can also be separated directly by flow cytometry, and the separated cells can be cloned for further study.

(4) Detection of relative activity of antigen-specific T cells

The interaction between TCR and MHC molecules plays an important role in the formation, activation, proliferation, function, peripheral tolerance and elimination of T cell phenotypes. By blocking the binding of MHC-tetramer to TCR, it is helpful to understand the difference of TCR affinity between different T cells.

(5) In situ tetramer labeling

In situ tetramer (IST) method is to use MHC-tetramer directly to label antigen-specific T cells in tissue sections. The method is divided into direct IST and indirect IST. The difference between them is that the indirect method reacts with the antibody of the marker linked to MHC-tetramer on the basis of the original MHC-tetramer labeled cells, thus further amplifying the labeling signal of the tetramer-positive cells. This method can compare the number, subtypes and functions of antigen-specific T cells in various tissues, and further combine with in situ hybridization (ISH), which is helpful to understand the natural state of immune response in organisms. By subcellular localization of TCR and related molecules, we can obtain the cellular biological characteristics of T cells in vivo. Responses and connections to different stimuli.

Advantages and limitations of MHC Tetramer

MHC Tetramer avoids the complicated process of in vitro culture, specific proliferation and isotope killing in conventional CTL detection. By synthesizing MHC Tetramer, flow cytometry can be used to detect antigen-specific CTL simply and accurately, which can be used for direct qualitative analysis and quantitative detection of antigen-specific CTL, and specific CTL can be separated from lymphocyte population for further study. Further research.

MHC Tetramers also have their own limitations. First of all, it must have a clear antigen epitope, and the antigen epitope it uses is a single epitope, there are often many antigens in the state of disease, and each antigen has many T cell recognition antigen epitopes. Therefore, it is necessary to study each antigen epitope at the same time in the study. Secondly, human MHC-I molecules express differently in different races and have polymorphism, which hinders the unified use of tetramer reagents. In the study, tetramers of different HLA subtypes need to be prepared according to the research object. Thirdly, tetramer staining is based on the specific cell structure as the research object. It is necessary to further study the cell population and function objectively. In addition, the preparation process of MHC Tetramer is complex, the protein renaturation efficiency is low and the price is expensive, which limits its large-scale application to a certain extent.

Application Prospect of MHC Tetramer

Tetramer complex system is a new milestone in the research of CTL cell immune function technology. Although there are some limitations, more and more researchers are engaged in the construction of various types of tetramer complex. The transformation of MHC Tetramer is also under way. With the further development and improvement of this technology, it is expected that this technology will be more extensive and in-depth.

Application prospects.

Firstly, the application of this technology in epidemiological investigation, pathogenesis and antiviral treatment of viral diseases will open up a new prospect for the diagnosis and treatment of viral diseases. Secondly, with the establishment of various tumor specific antigens, tetramer assay has great potential in the early diagnosis of some tumors. Quantitative screening of tumor-specific CTLs for in vitro culture and amplification will greatly promote the development of cancer cell immunotherapy. In addition, the diagnosis and treatment of intracellular microorganisms, pathogenesis, diagnosis and treatment of autoimmune diseases have great potential for development.