Fig 1: XCL1(CC3)-LPETG retains binding specificity to XCR1 after site-specific labeling with a small fluorophore. (A) Representation of 6×His-SUMO-XCL1-LPETGG-FLAG produced in BL21(DE3), site-specific labeling via sortase-mediated transpeptidation, and structure of XCL1(CC3) after purification and refolding. V21C and V59C point mutations stabilizing the a-ß chemokine fold are highlighted in green, and two cysteine residues C11 and C48 present in the native XCL1 are highlighted in blue. Adapted from 2HDM structure20 and modeled in YASARA. (B) Production of XCL1(CC3) in BL21(DE3) Escherichia coli. ~30 kDa 6×His-SUMO-XCL1(CC3)-LPETGG-FLAG is present in bacterial lysate (lane 1) and enriched by pooling the soluble and insoluble fractions (S+I, Lane 2). 6×His-SUMO-XCL1 is eluted (lane 3) after nickel affinity purification, and solubility tag digested (ULP1, lane 4). XCL1(CC3) is isolated after a second nickel purification (FT Ni2+, Lane 5) with high purity. (C) Overlay of Sypro staining and 488 nm in-gel fluorescence. Site-specific labeling of XCL1(CC3) (lane 1) with 3M eSrtA (lane 2) and GGGCK(FITC). After reaction (lane 3), XCL1(CC3)-FITC is purified by nickel affinity purification (FT Ni2+, Lane 4), FITC excess removed by PD-10 desalting (Lane 5) and pure product is obtained after concentration (Lane 6). (D) Commercial anti-XCR1-PE and XCL1(CC3)-FITC identify a comparable subpopulation of ~60% CD141+ cDC1s without staining CD141- cDC2s, confirming that XCL1(CC3)-FITC specifically binds to XCR1. (E) Surface staining of cDC1s with 10 µg·mL-1 XCL1(CC3)-Cy5.5 or sdAb-Cy5.5 at 4°C for 30 min before fixation shows specificity of XCL1(CC3) for XCR1, as imaged by confocal microscopy. cDC1, conventional dendritic cell type 1.
Fig 2: PEGylation of GGG-K(N3)-S7Abu enables its conjugation to XCL1(CC3). (A) Site-specific PEGylation of GGG-K(N3)-S7Abu with DBCO-PEG5k. (B) 3M sortase-mediated site-specific labeling of XCL1(CC3) (lane 1) with GGG-K(PEG)-S7Abu allows product formation (lane 4). Sortase is removed by incubation with Ni-NTA resin (lane 5), excess of nucleophile and PEG5k is removed after cation exchange (lane 6), and unreacted XCL1(CC3) removed by incubation with anti-FLAG beads, allowing isolation of pure XCL1(CC3)-K(PEG)-S7Abu (lane 7). 3M eSrtA (lane 2) activity is confirmed by hydrolysis of XCL1(CC3) in absence of peptide (lane 3). Densitometry was performed to calculate the concentration of XCL1(CC3)-K(PEG)-S7Abu used in cell experiments. (C) Incubation of XCR1-expressing cDC1s with anti-XCR1 in presence or absence of XCL1(CC3)-K(PEG)-S7Abu shows that the PEGylated vaccine retains binding to XCR1. (D) cDC1 migration towards media (Ø), 10 ng·mL-1 rhXCL1 (+, BioLegend), XCL1(CC3), and XCL1(CC3)-K(PEG)-S7Abu, shows that modification of XCL1 does not impair its chemotactic activity. N=4 independent donors, normalized to rhXCL1. One-way ANOVA, ***p<0.001. (E-G) 5M sortase-mediated site-specific labeling of XCL1(CC3) (lane 1) with GGG-K(PEG)-Y7A (gp100) (E), GGG-K(PEG)-R7A (TGFßRII) (F) and GGG-K(PEG)-F7M (caspase 5) (G) allows product formation (lane 4). Sortase is removed by incubation with Ni-NTA resin (lane 5), and excess of nucleophile and PEG5k is removed after cation exchange (lane 6), allowing isolation of pure XCL1(CC3)-K(PEG)-S7Abu (lane 6). 5M eSrtA (lane 2) activity is confirmed by hydrolysis of XCL1(CC3) in absence of peptide (lane 3). ANOVA, analysis of variance.
Fig 3: cDC1s treated with XCL1(CC3)-K(PEG)-S7Abu cross present S7Abu to CD8+ T cells following XCR1-mediated uptake. (A) Experimental setup allowing for cdc1 isolation and CD8+ T cell transfection with S7Abu-specific TCR. (B, C, D) increased activation of CD8+ T cells through XCR1 targeting by cDC1s treated with XCL1(CC3)-K(PEG)-S7Abu (0.1 and 1 µM) compared with vehicle control, as measured by CD8+ T cell division index (B), expression of activation markers (CD25, PD-1) and downregulation of TIGIT (C), and IL-2 and IFN? secretion in the culture media (D). N=3 independent donors. One-way ANOVA with Tukey’s post hoc correction for multiple testing, *p<0.05, **p<0.01. ANOVA, analysis of variance; ns, not significant.
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