Part:BBa_K4040002

FcRγ Function Unit

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 799
  Illegal NgoMIV site found at 958
  Illegal NgoMIV site found at 1024
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 679
  Illegal BsaI.rc site found at 1240
  Illegal BsaI.rc site found at 1249
  Illegal SapI.rc site found at 1197
  

Usage and Biology

FCER1G is an adapter protein containing an immunoreceptor tyrosine-based activation motif (ITAM) that transduces activation signals from various immunoreceptors. As a component of the high-affinity immunoglobulin E (IgE) receptor, it mediates allergic inflammatory signaling in mast cells. As a constitutive component of interleukin-3 receptor complex, it selectively mediates interleukin 4/IL4 production by basophils, priming T-cells toward effector T-helper 2 subset. It associates with pattern recognition receptors CLEC4D and CLEC4E to form a functional signaling complex in myeloid cells. Binding of mycobacterial trehalose 6,6'-dimycolate (TDM) to this receptor complex leads to phosphorylation of ITAM, triggering activation of SYK, CARD9 and NF-kappa-B, consequently driving maturation of antigen-presenting cells and shaping antigen-specific priming of T-cells toward effector T-helper 1 and T-helper 17 cell subtypes. FCER1G may also function cooperatively with other activating receptors. It is functionally linked to integrin beta-2/ITGB2-mediated neutrophil activation, which is also involved in integrin alpha-2/ITGA2-mediated platelet activation.

Figure 1. Structure of FCER1G.

Background and detail description

Used in our project

To program engulfment based on recognition of the SARS-CoV-2 spike protein, we used a CAR design for the synthetic receptor strategy in our study. The synthetic receptors were constructed to contain an scFv derived from an antibody recognizing the virus spike protein, CR3022, which has been reported to bind with the receptor-binding domain of the SARS-CoV-2 S glycoprotein with high affinity, and the CD8 transmembrane domain present in the aCD19 CAR for T cells [1]. For the cytoplasmic domains, we used the common g subunit of Fc receptors (CARg), MEGF10 (CARMEGF10), MERTK (CARMERTK) and CD3z (CARz) in our study(Figure 2A).These cytoplasmic domains are capable of promoting phagocytosis by macrophages.

Next, we used lentiviral vector technology to express the fusion constructs in human macrophage THP-1 cells using clinically validated techniques [2]. The cDNA sequences containing the various fusion constructs were cloned into a third-generation lentiviral vector in which the CMV promoter was replaced with the EF-1a promoter [3]. An extracellular MYC epitope was cloned into the receptors to permit detection by flow cytometry. Lentiviral vector supernatants transduced THP-1 cells with high efficiency (Figure 2B). The phagocytic potential of human macrophage THP-1 cell lines expressing different CAR receptors or a truncated CAR receptor (CARD) lacking the intracellular domain was measured with a cell-based assay. Consistent with previous reports [2, 4], CAR macrophages and control untransduced (UTD) macrophages did not show notable phagocytosis of 293 cells; however, CARMEGF10, CARg and CARz cells but not CARMERTK,CARD, or UTD macrophages phagocytosed Spike-bearing 293 cells in an S-specific manner (Figure 2C). CAR-mediated macrophage phagocytosis was further confirmed by a luciferase-based killing assay, and our data showed that CARMEGF10, CARg and CARz cells eradicated S protein-expressing 293T cells in an antigen-specific manner(Figure 2D).

Antibody-mediated phagocytosis and internalization of virions are important mechanisms of antiviral activity performed by macrophages against pathogens; however, using the phagocytosis assay developed for SARS-CoV-2, we observed low levels of phagocytic activity when UTD cells directly contacted virions. Phagocytic activity was not significantly increased when CARD cells rather than UTD macrophageswere the phagocytes in the assay, suggesting that the extracellular domain of the CAR alone is not sufficient to induce strong virion internalization. CARg, CARMEGF10, and CARz mediated similar significantly stronger levels of SARS-CoV-2 phagocytosis by THP-1 cells than CARD.We observed not only strongly increased induction of IL-6, IL-8 and TNF-a but also induction of IFN-g and IL-10 in SARS-CoV-2-treated CARg and CARz cells.

CARD cells showed no protective effect in the infection assay, although they had a similar capacity to bind to the S protein, suggesting that the intracellular signaling domain is necessary for virion clearance by CAR macrophages.[5]

Figure 2. CARs mediate phagocytosis of SARS-CoV-2 virions. (A) Different cell lines were inoculated with a SARS-CoV-2 pseudotyped virus. At 16 h post inoculation, pseudotyped virus entry was analyzed by determining the luciferase activity in cell lysates. Signals obtained for particles bearing no envelope protein were used for normalization. The average of three independent experiments is shown. Error bars indicate the SEM. (B) The uptake of pseudotyped virions by UTD and CAR macrophages was analyzed by flow cytometry. Different cell lines were stained with an anti-S primary Ab. The histograms shown in black correspond to the isotype controls, whereas the red histograms indicate positive fluorescence. Data are reported as the phagocytic score (% positive cells x MFI, right panel). (C) Cell lines were infected with the SARS-CoV-2 pseudotyped virus or mock infected. Cytokine levels in the supernatants were determined by a multiplex bead array. The relative level was calculated as the ratio of the infected cells to the mock-infected THP-1 cells. Data are shown as the mean ± s.d.

Used for a CAR

Macrophages expressing a CAR-P with the Megf10 (CARPMegf10) or FcRgamma (CAR-PFcRgamma) intracellular domain promoted significant engulfment of CD19 beads compared to macrophages with no CAR (Figure 3,Figure 4)[6].

Figure 3. J774A.1 macrophages expressing aCD19 CAR-P with the indicated intracellular signaling domain (green) engulf 5 mm silica beads covered with a supported lipid bilayer containing His-tagged CD19 extracellular domain. The beads were visualized with atto390-labeled lipid incorporated into the supported lipid bilayer (magenta). Cells infected with the cell membrane marker, mCherry-CAAX, were used as a control (no CAR, top left). To the right of each image is a histogram depicting the frequency of cells engulfing the indicated number of beads.

Figure 4. The average number of beads eaten per cell.

CAR-P promotes trogocytosis and whole cell eating(Figure 5)[6]. Previous work demonstrated that PI3K signaling is important for internalization of large targets (Schlam et al., 2015).

Figure 5.(A) J774A.1 macrophages expressing the aCD19 CAR-PMegf10(top panel, green in merge, left; greyscale, center) engulf pieces of CD19 +Raji B cells (labeled with mCherry-CAAX; magenta in merge, left; greyscale, right). The corresponding control aCD19 CAR-PGFP-infected cells are shown below. Arrows point to pieces of ingested Raji B cell. The proportion of CAR-P expressing macrophages internalizing one or more bite within 90 min is quantified on the right. Bites are defined as a fully internalized mCherry-positive vesicle >1 mm in diameter; n = 46 CAR-PMegf10macrophages, n = 39 CAR-PFcRVmacrophages and 102 CAR-PGFPmacrophages acquired during three separate experiments. (B) Time course of a J774A.1 macrophage expressing CAR-PFcRV(green) internalizing a whole Raji B cell labeled with mCherry-CAAX (magenta). These images correspond to frames from Figure 4—video 2. (C) Schematic shows the structure of CAR-Ptandemconstruct, combining the intracellular signaling domain from FcRV and the p85 recruitment domain from CD19. (D) Time course of a J774A.1 macrophage expressing CAR-Ptandem(green) internalizing a whole Raji B cell labeled with mCherry-CAAX (magenta). These images correspond to frames from Figure 4—video 3.The number of Rajis was then quantified by FACS. 2–3 technical replicates were acquired each day on three separate days. The number of Rajis in each replicate was normalized to the average number present in the GFP-CAAX macrophage wells on that day. * indicates p<0.01, *** indicates p<0.0001 by two-tailed Fisher Exact Test(a and e) or by Ordinary one way ANOVA with Dunnet’s correction for multiple comparisons (f); error bars denote 95% confidence intervals.

References

[1]Klichinsky M, Ruella M, Shestova O, Lu X, Best A, Zeeman M, et al. Human Chimeric Antigen Receptor Macrophages for Cancer Immunotherapy. Nat Biotechnol (2020) 1–7. doi: 10.1038/s41587-020-0462-y

[2]Levine BL, Kessler DA, Rappel WJ. Gene Transfer in Humans Using a Conditionally Replicating Lentiviral Vector. Proc Natl Acad Sci (2006)103:17372–7. doi: 10.1073/pnas.0608138103

[3]Fu W, Lei C, Liu S, Cui Y, Wang C, Qian K, et al. CAR Exosomes Derived From Effector CAR-T Cells Have Potent Antitumour Effects and Low Toxicity. Nat Commun (2019) 10:1–12. doi: 10.1038/s41467-019-12321-3

[4]Morrissey MA, et al. Chimeric Antigen Receptors That Trigger Phagocytosis. Elife (2018) 7:e36688. doi: 10.7554/eLife.36688

[5]Fu W, Lei C, Ma Z, Qian K, Li T, Zhao J, Hu S. CAR Macrophages for SARS-CoV-2 Immunotherapy. Front Immunol. 2021 Jul 23;12:669103. doi: 10.3389/fimmu.2021.669103. PMID: 34367135; PMCID: PMC8343226.

[6]Morrissey MA, Williamson AP, Steinbach AM, Roberts EW, Kern N, Headley MB, Vale RD. Chimeric antigen receptors that trigger phagocytosis. Elife. 2018 Jun 4;7:e36688. doi: 10.7554/eLife.36688. PMID: 29862966; PMCID: PMC6008046.