CAR-T细胞 z-bo 2020-09-30 296

　　The clinical application of transgenic T cells (CAR-T cells) expressing chimeric antigen receptors (CAR) in the treatment of B-cell malignancies has been successful, but cytokine release syndrome (CRS) prevents this situation. The effect of treatment on patients. It is well known that CRS is caused by an acute inflammatory reaction, which is characterized by fever, hypotension and respiratory failure related to elevated serum cytokines. Although it is reported that macrophages are involved in the etiology of CRS in a humanized mouse model treated with CAR-T cells, the mechanism leading to CRS is still unclear. The CAR-T cells injected into the patient will undergo activation and proliferation, and the rapidly proliferating CAR-T cells will cause the rapid mass death of B leukemia cells in a short time. Coincidentally, the disease burden of patients with acute lymphoblastic leukemia (ALL) is also closely related to the incidence and severity of CRS. It is unclear how this large-scale malignant B cell death is related to the pathogenesis of CRS.

　　Cells experience different types of death. Initially, dense population was the only regulated form of procedural death. However, recent studies have identified previously unrecognized programmed necrosis, which is characterized by rapid cell expansion, large bubbles on the progenitor cell membrane, and the release of pro-inflammatory factors. At least two programmed necrotic cell death pathways have been identified, including MLKL-mediated necrosis and GSMDD (GasderminD) or GSDME (GasderminE)-mediated apoptosis. Recruitment of RIPK1 to the TNF-α receptor forms a death complex with RIP3, which produces membrane nanopores, which can lead to necrosis. Unlike MLKL, GSMDD or GSDME is activated by inflammatory caspase (caspase 1, caspase 4, caspase 5 and mouse caspase 11) or caspase 3. They produce oligomers that insert into cell membranes to form and mediate nanopores. Cells burn.

　　In a new study, researchers from the Chinese Academy of Medical Sciences, Zhengzhou University, Huazhong University of Science and Technology and Peking University proved that human B leukemia cells and other target tumor cells express sufficient amounts of GSDME. provide. GSDME is effectively activated by Caspase 3, and Caspase 3 is activated by Granzyme B released by CAR-T cells, leading to thermal degradation of target cells. Factors released by thermal degradation stimulate macrophages to produce pro-inflammatory cytokines. This may lead to CRS in patients receiving CAR-T cell therapy. These researchers found that CAR-T cells activate the caspase 3-GSDME pathway of B leukemia cells by releasing a large amount of perforin and granzyme B, thereby causing thermal degradation and subsequent CRS. It has been observed that CAR-T cells undergo a process of proliferation and reach a very high frequency of proliferation at specific points in the body. Therefore, in a relatively short period of time, most target cells may undergo thermal degradation, and activated macrophages will produce IL-6 and IL-1β through activated caspase 1, thereby triggering CRS. Have. The elucidation of this molecular mechanism provides insights into clinical observations related to the severity of CRS, the number of CAR-T cells treated and the load of B leukemia cells.

　　"Detects the expression of GSDME in human B leukemia cells, MCF-7 breast cancer cells and mouse B16 melanoma cells. It acts as a pore-forming protein, and its activation is potentially dangerous and may lead to cell death. According to reports, GSDME is not expressed in many tumor cell lines tested. Consistent with this, the promoter region of the GSDME gene shows a hypermethylated state. This indicates that the GSDME gene suffers from epigenetic silencing in the cell. GSDME is considered to be a tumor suppressor gene, which can induce cell death caused by the caspase 3 lysis program, so that tumor cells can silence the expression of GSDME, thereby evolving epigenetic means for tumorigenesis. It might be so. However, the high expression of GSDME in BSD leukemia cells and other tumor cells indicates that GSDME may play an alternative role in the formation of tumor cell pores. People are currently studying how GSDME expression overcomes hypermethylation regulation, and whether GSDME has conventional functions other than pore formation. An important finding in this study is that CAR-T cells release more perforin/granzyme B than untransfected natural T cells. The cytolytic effector molecules released by T cells depend on the activation of two signals: MHC antigen peptide-TCR (signal 1) and CD80/CD86-CD28 (signal 2). The complete activation of signal 2 depends on the activation strength of the TCR signal. The light chain kinase (LCK) activated by the TCR signal phosphorylates CD28 tyrosine residues. At the same time, LAT and SLP-76 activated by TCR signal phosphorylate and activate the main CD28 downstream signal molecule PLC-γ, while phosphatidylinositol 4,5-bisphosphate (PIP2) is diacylglycerol (DAG). And inositol 1,4,5-triphosphate (IP3). Based on the understanding of T cell activation and genetic engineering progress, these researchers designed a synthetic CAR targeting human T cells. The basic concept of CAR design is to connect single-chain variable region fragments (scFv) to the signal transduction module in CD3ζ cells to induce T cell activation after antigen binding. Currently, this modular structure extends from a single CD3ζ signal domain to CD3ζ-CD28, CD3ζ–4-1BB or CD3ζ–CD28–4-1BB signal domains, thereby simulating signal 1 and signal 2, in view of the relationship between CAR and its antigen The affinity of TCR can be 100 times higher than that between TCR and MHC peptide complex, so this superior affinity can be combined with costimulatory signals. CAR-T cells can release a large amount of perforin/granzyme B required for target cells to generate heat through CAR-T cells. Once in the cytoplasm, Granzyme B can cleave the inactive precursor of Caspase 3 (Procuspase 3) into an active form. Activated caspase 3 induces apoptosis or GSDME lysis, which causes thermal degradation through the formation of membrane pores. However, cells have the ability to quickly repair pores formed in the membrane of progenitor cells. Whether GSDME causes thermal degradation depends on the balance between film pore formation and film repair. Although the content of GSDME is the same, natural TCRCD8 + T cells cause only low levels of GSDME lysis, while CAR-T cells release high levels of perforin/granzyme B and activate GSDME. The lysis that occurs during pyrolysis is highly pro-inflammatory due to the release of cytoplasmic content rich in damage-related molecular pattern (DAMP) molecules. In this study, these researchers confirmed that the pyrolysis experienced by tumor cells causes the activation of macrophages caspase 1 and GSDMD, which leads to the release of large amounts of pro-inflammatory cytokines and the development of CRS. .. Among these pro-inflammatory cytokines, IL-6 and IL-1β are particularly important. Clinically, IL-6 neutralizing antibodies are widely used to prevent and/or treat CRS in patients receiving CAR-T cell therapy. As a multifaceted cytokine, IL-6 is mainly regulated by transcription factors such as NF-κB, AP-1 and STAT3. Pathogen-associated molecular pattern (PAMP) molecules (such as lipopolysaccharide (LPS)) and DAMP molecules (such as heat shock protein (HSP) and HMGB1) stimulate macrophages by activating these transcription factors to stimulate IL-6. Can be generated. These researchers found that HMGB1 exists in the supernatant of thermally degradable cells (cells that receive thermally decomposed cells), and directly activates the synthesis of IL-6 and IL-1β precursors in macrophages, and its release depends on cystatin. The activation of caspase 1, which is an inflammation-induced caspase strictly regulated by the matrix. The matrix small molecule NLRP3 is extensively activated by a variety of stimuli, such as microbial toxins, granules, crystals, β-amyloid aggregates and extracellular ATP. In this study, these researchers found that the supernatant of pyrrolidine cells contained ATP. Treatment or degradation of ATP with P2X7 receptor antagonists may interfere with the ability of the supernatant of Pyrophyta cells to activate macrophage caspase 1. With the help of the mouse CRS model they developed, they can further prevent the development of CRS by knocking out GSDME of target tumor cells, knocking out macrophages or inhibiting caspase 1/GSDMD. I confirmed it. Elucidating this molecular approach is essential to better understand the toxicity associated with CAR-T cell therapy. In 2018, Verena Staedtke and colleagues discovered that catecholamine blockers can prevent macrophages from releasing pro-inflammatory cytokines. Therefore, blocking the combination of GSDME and catecholamines can better treat CRS without reducing tumor clearance.

　　This study reported that CAR-T cells cause thermal degradation in target tumor cells through a GSDME-dependent pathway, while CAR-T cells cause other mediators of thermal degradation of target cells. There may be a way. Recent studies have reported that CAR-T cells may mobilize TNF-α to mediate this killing process, which may have nothing to do with Granzyme B and Perforin. Have. One possibility of this discovery is that CAR-T cells can use a two-step strategy to attack target cells. Granzyme B and perforin caused the first wave of killings. When the target cell escapes the first wave of attack, TNF-α starts the second wave of killing. This may also explain why thermal degradation can be prevented without significantly affecting the killing effect mediated by CAR-T cells. Current research shows that there is a mechanism difference between the types of cell death caused by CAR-T cells and natural TCR-T cells, which is changed by converting tumor cell death from thermal degradation to apoptosis.