Fig 2: 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 3: GM-CSF Promotes cDC1 Differentiation from CB and Non-mobilized Adult Blood CD34+ Cells(A–C) Expanded CD34+ CB cells were differentiated with FT7 on OP9_DLL1 feeder layer cells in the absence or presence of GM-CSF added at different time points.(A) Top: frequencies of live CD123+ and CD141+ cells in the CD14-CD206- gate. Bottom: frequencies of CD141+CLEC9A- and CD141+CLEC9A+ cells within the CD141+ gate shown on the top. Plots are from one donor (representative of 4). FT7+G, 1 ng/mL GM-CSF added to FT7 on days 0, 7, and 14 of differentiation.(B and C) Frequencies (top) and absolute numbers (bottom) of cDC1s (B) and pDCs (C) generated in the absence or presence of GM-CSF added at the indicated concentrations on days 0, 7, and 14 (w1–3), 7 and 14 (w2–3), or 14 (w3) of differentiation. Absolute numbers of cells obtained were calculated for an initial input of 104 CD34+ CB cells, taking into consideration both the expansion and differentiation phases. Data shown are from 4 experiments, each with a different donor (symbols), with mean ± SD shown across donors for each experimental condition. Statistics were performed comparing each condition of GM-CSF supplementation with the FT7 control culture.(D) Carboxyfluorescein diacetate succinimidyl ester (CFSE)-stained cells were differentiated with FT7 or FT7+G (0.25 ng/mL) and harvested over time to measure cell expansion. Each symbol represents an individual donor (D1, D2, and D3). The open versus closed formats represent different days of harvest (day [d]6, d12, and d18).(E) Mean CFSE fluorescent intensity at days 6 and 12 on total cells in the same differentiation cultures as shown in (D).(F) Fraction of live (PI-Annexin-V-) versus early apoptotic (PI-Annexin-V+), late apoptotic (PI+Annexin-V+), or necrotic (PI+Annexin-V-/low) cells in cultures over time, for the individual donors (symbols), which mean ±SD shown across donors for each day.(G) Frequency of XCR1- versus XCR1+ cells in the cDC1s generated in vitro from FST7-expanded CB CD34+ cells differentiated for 18 days on OP9_DLL1 feeders with FT7 or FT7+G. Data shown are from 2 experiments, each with 1–3 different donors (symbols), with mean ± SD shown across donors for each experimental condition.(H) Frequency (top) and total numbers (bottom) of CD123+CD45RA+ pDCs and CD141+CLEC9A+ cDC1s generated from 104 non-mobilized FST7-expanded CD34+ cell from adult peripheral blood differentiated on OP9+OP9_DLL1 feeder with FT7 or FT7+G. Data shown are from 3 experiments, each with 2 different donors (6 data points with their mena ± SD shown for each condition).

Fig 4: 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 5: 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.