Fig 1: Activation of the Complement Cascade in Presence of Ad5 and 9C12 Results in Deposition of C4b on the Ad5 Capsid(A) Western blot of C4 (a-chain) cleavage in NHS (top) or C1q-depleted serum (bottom) in the presence of Ad5 and 9C12-WT over 60 min. See also Figure S2E.(B) Experimental set up (left): Ad5-mCherry+9C12-WT were incubated with HBS++/serum for 1 h at 37°C. Ad5-GFP was incubated with HBS++. Ad5-mCherry was added to HeLa TRIM21 KO cells immediately followed by Ad5-GFP. Right: relative infection of Ad5-mCherry and Ad5-GFP. x axis labeling corresponds to the buffer/serum that was incubated with Ad5-mCherry.(C) Western blot of C4 (a-chain) cleavage in NHS, C1q-depleted serum, or C4-depleted serum in the presence of Ad5 and 9C12-WT as indicated.(D) Elisa for C4 using serum (left) or purified protein (right). Ad5+9C12-WT were incubated with the indicated serum or purified protein and pelleted over a sucrose gradient.Error bars depict mean + SD of three replicates acquired in one representative experiment (A and D). Original western blots are included in Figure S6.
Fig 2: Expression of complement genes in iPSC-derived neural progenitor cells, neurons and astrocytes. Relative transcript levels of key complement system components, receptors, and regulators measured by RT-qPCR in: (A) iPSC-derived NPCs of control (n = 4) and ASD (n = 7) subjects; (B) iPSC-derived neurons of control (n = 3) and ASD (n = 5) subjects; (C) iPSC-derived astrocytes of control (n = 4) and ASD (n = 5) subjects. A significant decrease in the expression levels of C4A/B and SERPING1 mRNAs was observed in astrocytes derived from individuals with ASD compared to control astrocytes. ** p < 0.01.
Fig 3: GZMK activates the entire complement cascade.a. Model for GZMK-mediated complement activation. GZMK-expressing CD8+ T cells constitutively release GZMK in the absence of TCR stimulation. Secreted GZMK can bind plasma membranes likely by interacting with heparan sulfate glycosaminoglycans, where it cleaves C4 and C2 generating C4b and C2a. Due to its close proximity to the membrane, newly-cleaved C4b molecules can covalently bind membranes through their exposed thioester, associate with C2a, and form membrane-bound C3 convertases. These C3 convertases can cleave C3 into C3a and C3b. Nascent C3b molecules can opsonize target cells or associate with membrane-bound C3 convertases to form C5 convertases that can cleave C5 into C5a and C5b. C5b molecules associate with C6, C7, C8, and C9 to form a membrane attack complex (MAC) or terminal complement complex (TCC). b. Comparison between the alternative, classical, lectin, and GZMK-mediated pathways of complement activation. Complement activation occurs most efficiently on membranes. Soluble pattern recognition receptors like C1q and mannose-binding lectin initiate this process after recognizing danger signals on a surface. This first step results in the activation of initiator proteases like C1s and MASP1, which cleave the complement components C4 and C2. GZMK can direct complement activation to surfaces independently of soluble pattern recognition receptors due to its intrinsic ability to bind negatively charged molecules such as heparan sulfate glycosaminoglycans. Much like C1s, MASP1/2, and CFD, GZMK acts as an initiator protease that cleaves C4 and C2 into C4b and C2a. In the second step, C4b and C2a assemble at the membrane to form active C3 convertases that cleave C3 into C3a and C3b. In the third step, nascent C3b molecules join pre-existing C3 convertases to form C5 convertases that cleave C5 into C5a and C5b, with the latter associating with C6–9 to assemble the TCC/MAC.
Fig 4: GZMK cleaves the complement components C4 and C2 to generate a C3 convertase that cleaves C3 into C3a and C3b.a, Bulk CD8+ T cells were MACS sorted from peripheral blood of two donors and either left unstimulated or stimulated with anti-CD3/CD28 dynabeads for 6, 24, or 48 hours. Precipitated supernatants and lysates were analyzed by immunoblot using antibodies against GZMK and GZMB. b, Increasing concentrations of active GZMK or GZMA were incubated with serum-purified C4 for 4 hours and cleavage products were analyzed by immunoblot. Active C1s was used as a positive control for C4 cleavage into C4b. Purified C4b was used to confirm the size of the C4b fragment generated by C1s and GZMK. c, Active GZMK was incubated with C2 for 4 hours in the presence or absence of increasing concentrations of C4 and cleavage products were analyzed by immunoblot. Active C1s was used as a positive control for C2 cleavage into C2a. d,e, Increasing concentrations of active GZMK or GZMA were incubated with C2 + C3 + C4 and cleavage products were analyzed by immunoblot. Active C1s was used as a positive control for generation of a C3 convertase that cleaves C3 into (d) C3a and (e) C3b. Serum-purified C3a and C3b were used to confirm the identity of the fragments generated by C1s and GZMK, while purified iC3b was used to determine whether GZMK generates inactive C3b. Schematic representation of the assays are shown for b-e. Data are representative of at least four independent experiments.
Fig 5: GZMK triggers activation of the entire complement cascade.a, LAD2 mast cells were incubated with C3, C3a, or the reaction products obtained after incubating C1s or GZMK with C2 + C3 + C4. Degranulation was then assessed by flow cytometric staining of surface LAMP-1. b, HUVEC cells, synovial fibroblasts, or THP-1 monocytes were incubated with recombinant GZMK for 1 hour on ice, and surface-bound GZMK was then measured by flow cytometry after staining of unfixed, unpermeabilized cells. c, Surface deposition of C3b on HUVEC cells after incubation with C2 + C3 + C4 with or without C1s, GZMK, or GZMA, as measured by flow cytometry. Normal human serum (NHS) was used as a positive control. d, GZMK surface binding (left) and C3b deposition (right) on HUVEC cells after incubation with C2 + C3 + C4 with or without GZMK in the presence or absence of heparin. NHS was used as a control. e, GZMK either pre-bound to the surface of HUVECs or in solution without HUVECs was incubated with C2 + C3 + C4 for 0.5, 1, 2 or 4 h and the supernatants were analyzed by immunoblot for generation of C4a, C2b, and C3a. Purified C4a, C2b, and C3a were used to confirm the size of the fragments generated by GZMK. f, HUVEC cells were incubated with C2 + C3 + C4 + C5 with or without GZMA or GZMK and the generation of C5a was then measured by ELISA. g, C5aR1-expressing Chem-1 reporter cells labeled with Fluo-5F were incubated with C5, C5a, or the supernatants obtained after incubating HUVEC cells with C2 + C3 + C4 + C5 with or without GZMA or GZMK. Calcium flux was immediately assessed by flow cytometry and is presented as MFI (MFI minus unstimulated MFI). h, HUVEC cells were incubated with C2 + C3 + C4 + C5 + C6 + C7 + C8 + C9 with increasing concentrations of GZMK or GZMA. C5b,6–9 was used as a positive control. Terminal complement complex formation was then measured by flow cytometry. Histograms depict representative data. i, Surface deposition of C3b (left) and C4d (middle), and TCC formation (right) on HUVEC cells after incubation with C1q-depleted serum and increasing concentrations of GZMK, as measured by flow cytometry. NHS was used as a positive control. Data in a-i are representative of at least 3 independent experiments. P values were calculated using (c, f-i) one-way analysis of variance (ANOVA) with Dunnett’s multiple comparisons tests or (d) a two-way ANOVA with Sidak’s multiple comparisons tests. *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001, NS = not significant. Data are mean ± s.d. of (c,d,f,h) three or (i) five independent experiments or (g) three technical replicates from a representative of three independent experiments. (b) Data are representative of three independent experiments.
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