Research progress in the application of MHC tetramer technology (I)

Posted by beauty33 on May 19th, 2021

T cells aggregate to form soluble peptide-MHC tetramers to increase the sensitivity of specific T cell detection through the T cell receptor (TCR) and MHC molecular recognition and binding mechanism to bind peptide major histocompatibility complex (pMHC) ligands on the surface of T cells, which is tetramer technology. MHC tetramer technology can effectively detect antigen-specific T cells, and plays an important role in tumor antigens, antiviral infection and specific T cell research.

1. The molecular structure basis and basic principles of MHC tetramer technology

Immune recognition is the basis for the body's immune system to produce specific immunity. Through the specific recognition of foreign antigens, the body produces a corresponding immune response, and finally effector cells or effector molecules with certain biological activity eliminate pathogenic substances. MHC molecules participate in the processing, processing and presentation of antigens, forming MHC-peptide complexes (pMHC), which are the basis of cellular immune responses. Tetramer technology is based on TCR, through the APC/target cell surface pMHC molecules to form a "TCR-pMHC" triad or tri-molecule complex structure, thereby activating T cells.

MHC class Ⅰ molecules recognize TCR and activate CD8 + T cells, and MHC class Ⅱ molecules recognize TCR and activate CD4 + T cells. After being activated, T cells further differentiate to form effector T cells, which play a corresponding role. Studies have shown that MHC class II molecules are heterodimers composed of α and β chains. The basic structures of the two polypeptide chains are similar. The N-terminus is outside the cell, the C-terminus is inside the cell, and the extracellular part occupies the entire chain. 2/3 of. Both the alpha chain and the beta chain extracellular sites can be subdivided into 2 domains each containing 90 amino acid residues, called α1, α2, β1, and β2, which are encoded by MHC-II gene exons 2 and 3, respectively Among them, α1 and β1 constitute the peptide-binding groove, and the polymorphic residues of class II molecules are mainly concentrated in α1 and β1. Like class I molecules, the polymorphism of MHC class II genes determines the biochemical characteristics of their peptide binding grooves, as well as the selectivity and affinity of MHC class II molecules to bind antigen peptides and be recognized by T cells. α2 and β2 belong to the Ig gene superfamily. During the antigen presentation process, the binding site of CD4 molecules on the surface of helper T cells (Th) and MHC class II molecules is located in the immunoglobulin (Ig)-like Β2 domain of non-polymorphic region. The transmembrane region contains 25 amino acid residues, and the formed dipeptide chain is coiled into a helix, connected to the extracellular part by means of a short hydrophobic region, and fixes the entire polypeptide chain to the cell membrane. The C-terminus of class II molecules is free in the cytoplasm and contains 10-15 amino acid residues, while the intracellular region is mainly involved in transmembrane signal transmission.

Current studies have shown that there are often some conserved amino acid residues at both ends of the antigen peptide binding groove, which can form a stable hydrogen bond net (HDN) with the N-terminal and C-terminal of the antigen peptide, and bind to MHC as The antigenic peptides of the complex often carry 2 or more amino acid residues that specifically bind to the peptide binding groove of the MHC molecule, called anchor sites, and these corresponding amino acids are also called anchor residues. The position of the anchor residue in the peptide binding groove of the MHC molecule forms a pocket, also known as anchoring. The differences in the amino acid structure of different MHC molecules are mainly reflected in the different pocket sizes, shapes and charges, and therefore determine the antigen peptides that a specific MHC molecule can bind. The binding of antigen peptides to MHC molecules is relatively selective. The composition of anchor residues of antigen peptides that bind to specific MHC molecules is called sequence motifs. Compared with MHC class I molecules, the peptide binding grooves of class II molecules are more open at both ends, which can accommodate peptides containing 13-25 amino acid residues (or longer), but they are usually enzymatically digested to contain 13~ A peptide of 17 amino acid residues. Because the peptide binding grooves of class II molecules have greater compatibility, it is difficult to analyze the anchor residues of the antigen peptides to which they bind. The signal of T cell recognition of antigen is mainly activated by TCR recognizing specific pMHC. TCR and CD3 are both characteristic surface markers of mature T cells. Most T cells involved in the immune response express TCRαβ on the surface, which is non-covalently bound to the CD3 molecule to form a complete TCR-CD3 complex, and jointly perform the recognition and signal transmission of the pMHC molecular complex on the APC surface. At the same time, this process It also requires the participation of CD4 or CD8 (co-receptor) on the surface of T cells. CD4 or CD8 can respectively bind to MHC II or MHC class I molecules on the surface of APC (or target cells), thereby enhancing the affinity of TCR and specific pMHC, and significantly enhancing the sensitivity of T cells to antigen stimulation. In summary, the tetramer technology is formed based on this molecular structure.

In order to meet the analysis needs of antigen-specific CD8 + T cells, especially in vitro studies, there have been studies designed to design biotin-labeled reversible polymers, namely the reversible polypeptide nitrilotriacetic acid polymers (NTAmers). This technology is designed using the principle that a single Ni2 + -NTA complex and oligohistidine (His) reversibly combine to form a multivalent Ni2 + -NTAHis complex. Poly NTA is combined with biotin (labeling molecule), histidine is labeled with pMHC molecule, NTA and histidine are combined to form a multivalent Ni2 + -NTA-His complex with a stable structure, making the tetrameric complex very stable. In addition, NTAmers multimers can be quickly dissociated in the presence of imidazole. Based on this feature, this method can separate cells and perform human leukocyte antigen-A2 (HLA-A2) polypeptide monomers and T cells. Off-kinetics research. Batard et al. developed dextramers, which used dextran molecules to couple multiple polypeptide-MHC class I molecular complexes, and labeled dextran with a fluorescent dye PE. In addition, conditioned MHC ligands have been developed, which can synthesize more stable MHC complex molecules, but such molecules can be decomposed under ultraviolet irradiation. Therefore, this method can control the selective binding of MHC molecules to polypeptide molecules under natural light conditions, and can be used for multimer combinatorial staining to detect multiple parameters in parallel. According to the metal molecule-labeled polypeptide MHC molecule, it can be used in mass spectrometry flow cytometry CyTOF (cytometry by time-of-flight), which can more accurately define the antigen specificity of T cells than flow cytometry.

To be continued in Part II…