Cell: Heavyweight, Chinese and foreign team jointly issued, is expected to design a new CAR-T therapy.

On July 29, 2020, Huang Chaolan, a team at the Center for Precision Medicine Multi-Group Research at Peking University's Department of Medicine, the Shanghai Institute of Biochemistry and Cell Research of the Chinese Academy of Sciences, and the HuiEnfu team at the University of California, San Diego, jointly published a paper on Cell entitled "Multiple Signalings of CD3and and Application its in CAR-Tsrapy The" The new method of absolute quantitative proteomics of mass spectrometry reveals the full picture of the dynamic phosphorylation modification of the T cell receptor-coreceptor (TCR-CD3) complex, analyzes the mystery of the phosphorylation characteristics of different CD3 chain ITAM domains, and finds one of the new functions of monophosphate of the sub-CD3, which is expected to help design a new CAR-T therapy.
TCR-CD3 complex plays a decisive role in the development, activation and immune response of T cells.
this important role comes from the immunoreceptor tyrosine activation base sequence (Betareceptortyrosine-based activation-itAM) at the inner end of the CD3 chain cell.
and the diversity of ITAM functions depend mainly on tyrosine phosphorylation in its domain, such as recruiting SYK kinase family protein ZAP70 to activate downstream signal conduction.
, the functionality of ITAM is widely used in the study of chimeric antigen receptors (CARs).
, CD3-sub-chains are often used to build CAR-T cell therapy anti-tumor activity, but the function of other CD3 chains and whether they can be applied to CAR design has yet to be further developed.
in-depth exploration of CD3 ITAM's tyrosine dynamic phosphating model provides core information for a comprehensive understanding of the functions of different CD3 chains.
The TCR-CD3 receptor complex has 10 ITAM domains with 20 phosphorylation sites, and it is technically challenging to achieve quantitative analysis of all phosphorylation sites at time resolution.
in order to visually compare the phosphorylation patterns of different TCR stimuli and accurately map the dynamic process of tCR-all tyrosine phosphorylation, the Huang Chaolan team developed a novel absolute quantitative method, Targeted-IP-Multiplex-Light-Absolute-Quantitative Mass Spectrometry (TIMLAQ-MS).
the method differs from the currently reported absolute quantitative method of proteomics, without the need to add isotope relabeled synthetic peptide segments, but cleverly uses the series mass label (TMT) to design the mixing of six standard samples and four analytical samples as an internal standard.
standard samples are a mixture of synthetic non-remarker phosphorylation/non-phosphate CD3 peptides (A) and background proteins (B) immunocipitated by IgG antibodies from unanti-antigen-stimulated T cells; PRM) method to obtain antigen stimulation, TCR-CD3 immunoprecipitation (IP) complex in different tyrosine sites phosphorylation / non-phosphorylation at different point in time quantitative results.
TIMLAQ successfully bypassed the isotope relabeled peptides commonly used in previous quantitative methods, not only saved costs, but also effectively reduced the complexity of the method and data acquisition errors, further improved the quantitative accuracy, and finally fully realized the absolute quantification of all ITAM phosphorylation modifications at different points in a single measurement, depicting the TCR-CD3 complex of tyrosine dynamic phosphorylation modification.
used this method to identify the different TCR stimulation conditions, CD3 sub-bases are mainly shown as double phosphorylation modification mode, while CD3 is presented with a single phosphorylation modification mode.
previous studies have shown that bisphosphosphinized ITAM and kinase family protein ZAP70 have a strong binding and activate downstream signal conduction, while monophosphate ITAM shows a very low binding.
this particular new discovery in the new study, which will drive researchers to explore further the new potential function of CD3 in the TCR pathway.
results show that the CD3 of monophosphate can be reduced by special collection inhibitory Csk kinase to reduce The TCR signal conduction, indicating that tCR has both activated and inhibited elements, and overall presents as a self-made signal conduction mechanism.
further in-depth study by the author's team and found that once the CD3 cytoplasmic domain is integrated into the second generation CAR, the ITAM domain of CD3, can reduce the production of CAR-T cytokines by collecting Csk, while the BRS domain of CD3, which can promote the persistence of CAR-T cells by raising p85.
in general, the design of applying CD3 to CAR can significantly improve the anti-tumor activity of CAR-T cells. Professor
Huang Chaolan is director of the Center for Precision Medicine Multi-Group Research, Peking University Medical Department, Associate Professor of Basic Medicine, Peking University Medical Department, Research Fellow of the Joint Center for Life Sciences, Peking University, and Emeritus Professor, University of Manchester.
in recent years, Professor Huang Chaolan has led a team to develop new methods of proteomics based on mass spectrometry, and the laboratory has internationally leading instruments, technologies and methods dedicated to providing the most quality assurance of comprehensive proteomics and mass spectrometry techniques for the challenges encountered in biological and clinical research.
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