Each part of the CAR design, from the recognition domain to the ITAMs of CD3 can be optimized to enhance downstream signaling. of receptors and antigens, proximal signaling, immunological synapse formation, and late signaling outcomes. Signaling through different T cell subtypes and how signaling is translated into practice are also discussed. construct to the TCR -chain C-region ( em TRAC /em ) locus.130 Therefore, the timing, degree, and duration of CD28 signaling may be crucial for driving CD28 CAR T cell metabolism and differentiation. ICOS is upregulated upon T cell activation and boosts activity via PI3K signaling in a manner similar to CD28. However, its transmembrane domain is critical and sufficient to recruit Lck to augment CAR-T cell signaling.80 In addition, the ICOS intracellular domain has a different structure than the CD28 domain, leading to reduced IL-2 production, possibly Deoxycorticosterone due to the inability to recruit Grb2.131 In contrast to CD28, the ICOS costimulatory domain induces CAR-T cells toward Th1/Th17 polarization, with increased expression of IL-17A, IL-17F, IL-22, IFN, and T-bet. Because of this Th1/Th17 polarization, ICOS-based CAR-T cells had a significantly Rabbit Polyclonal to AurB/C higher subpopulation of persistent CD4+ CAR-T cells in vivo than either CD28- or 4-1BB-based CAR-T cells.132 TNF receptor superfamily signaling motifs The TNF receptor superfamily (TNFRSF) on T cells provides costimulatory or coinhibitory signals in T cell responses. For example, the TNF receptor TNFR2 on activated T cells induces a costimulatory signal for T cell proliferation and differentiation.133 In addition, 4-1BB (CD137, TNFRSF9) is transiently expressed on T cells upon TCR stimulation and provides a costimulatory signal for T cell proliferation,134 survival,135 effector function, and differentiation into memory T cells.136 Another member in this family, Herpes virus entry mediator (HVEM), is both a costimulatory receptor for TNF-like molecule LIGHT137 and a ligand for the inhibitory receptor BTLA.133 As the most commonly used TNF-receptor-related costimulatory domain in CARs, 4-1BB exploits TNFR-associated factor family members (TRAFs) to form the signalosome and promotes the nuclear translocation and transcriptional activity of NF-B via the activation of the IB kinase complex (IKK/). Although 4-1BB-CAR-T cells increase IL-2 and IFN secretion, not to the same extent as CD28 CARs.138 In addition, 4-1BB-CARs induce the upregulation of anti-apoptotic protein Bcl-xL, and most importantly, 4-1BB-CAR induces proliferation, Tcm cell differentiation, and increased fatty acid oxidation through increased mitochondrial biogenesis to a greater extent than is induced by CD28-CARs.127 Because of these characteristics, 4-1BB-CAR-T cells showed great potency in clinical settings.24 Nevertheless, long-term exposure to resistant cancer cells induces dysfunction-associated gene expression in 4-1BB-CAR-T cells. In fact, 4-1BB-CAR-T cells show enhanced promoter accessibility to several key Deoxycorticosterone immunosuppressive transcription factors ( em TOX2, IRF8 /em , and em PRDM1 /em ).139 In addition, the overexpression of 4-1BB-CAR in T cells can lead to a negative effect through the induction of apoptosis via the upregulation of ICAM-1 and Fas/FasL.140,141 This fratricide was observed during the ex vivo expansion of 4-1BB-CAR-T cells when CD5 was targeted. Other TNFRSF molecules have also been tested as costimulatory domains, although their impact and downstream signaling after integration into CARs have not yet been systematically studied. HVEM belongs to the TNFRSF family. It has been shown to increase T cell functions such as IL-2 production, oxidative phosphorylation, and glycolysis, although it is also involved in the coinhibitory network described above. HVEM, as a costimulatory domain in CAR, attenuated Deoxycorticosterone the exhaustion of CAR-T cells and induced a balanced central memory (Tcm) and effector memory (Tem) cell population.142 OX40 is another TNFRSF protein used as a costimulatory domain in constructed CARs. OX40 activates both the PI3K/AKT and NF-B signaling pathways. Interestingly, the addition of OX40 after the CD28 and Deoxycorticosterone CD3 domains diminished the production of IL-10 in the CD28-CAR-T cells, although the detailed mechanism is not well understood.143 Cytokine receptor protein domains in CARs The purpose of costimulatory domain integration is to provide Deoxycorticosterone signal 2 for T.