Age and gender-matched C57BL/6 mice were purchased from Envigo Laboratories (France). Batf3 knock out (KO) mice were bred in our facilities under specific pathogen-free conditions. All animal experimentation was performed according to ethical approval from the Canton de Vaud authorities, Switzerland. Veterinary authorization number VD2273.

DNA sequences were inserted into the expression vector pMP-PB (Excellgene) by In-Fusion technique (Clontech). DNA sequences are shown in Supplementary Figure 1. Positive clones were verified by DNA sequencing (Microsynth). Middle scale protein production was performed in Chinese Hamster Ovary (CHO) cells at the Laboratory of Cellular Biotechnology of EPFL, Lausanne, Switzerland. Xcl1 fusion proteins were purified from the supernatants of 7-day CHO cultures. Purification was performed by affinity chromatography using Protein A resin (GE Healthcare, cat no 17-1281-02). Proteins were eluted with Glycine 0.1 M pH 3.0 and dialyzed against PBS overnight. After confirming their size and purity by SDS-PAGE, recombinant proteins were passed through a Mustang Q membrane (PALL Corporation) for endotoxin removal. Commercial Xcl1 was purchased from Hölzel Diagnostika Handels GmbH, Germany (item n°50677-M08B).

Spleens from naïve WT (C57BL/6) and Batf3^−/− mice were enriched for CD11c⁺ cells using CD11c (N418) microbeads (cat number 130-052-001, Miltenyi Biotec). DC-enriched suspensions from spleens of WT or Batf3^−/− mice were incubated with purified Xcl1-(OVA SLP)-Fc and Xcl1-Fc fusion proteins at 37°C for 35 min. Cells were washed and binding of fusion proteins was assessed using PE-conjugated anti-mouse IgG1 antibody.

Spleens from naïve WT (C57BL/6) mice were enriched for CD11c⁺ cells using CD11c (N418) microbeads (cat number 130-052-001, Miltenyi Biotec). 1 x 10⁶ cells (CD11c⁺ DC purity of ~50%) were resuspended in 0.1 mL of chemotaxis medium (RPMI1640, 1% BSA, 50 μM ß-ME, 100 μg/mL penicillin/streptomycin) and added to the upper chamber of a 24-transwell plate (with 8 μm pore, Corning). In the lower chamber, 0.5 mL of chemotaxis medium was added, containing either 250 ng/mL of commercial Xcl1, or 1,000 ng/mL of Xcl1-(OVA SLP)-Fc or Xcl1-Fc fusion protein to have an equimolar concentration of Xcl1 of 25 nM. After incubation for 2 h at 37°C (5% CO₂), bottom chambers were flushed with ice-cold PBS containing 10 mM EDTA and DCs were analyzed by FACS. Cells were incubated for 5 min on ice with 2.4 G2 to block Fc receptors, Xcr1⁺ DCs were detected via incubation with Xcl1-Fc protein (19 nM) for 30 min at 37°C, followed by washing and staining with PE-conjugated anti-mouse IgG1 on ice for 30 min. Afterwards, surface markers antibodies were added in a mix, on ice, for 30 min. DCs were identified by first excluding CD3⁺ B220⁺ and CD11b⁺ cells and gating on CD11c⁺ CD8α⁺ cells.

Alexa-488 dye (DY-490-NHS-Ester, from Dyomics, product number 490-01) was resuspended in DMSO (the molar ratio between 1 mg of dye and 1 mg of the Xcl1 fusion proteins is 40.2, hence 40.2 μL of DMSO were added). The dye and the 10x reaction buffer (1 M Na Phosphate, 1.5 M NaCl, pH 7.1) were added to the fusion proteins at a volume ratio of 1:10, and mix was incubated at room temperature for 1.5 h in rotation and protected from light. Desalting columns (Zeba Spin desalting column, Thermo Scientific, product number 89,890) were washed with PBS by spinning 1,000 g for 2 min. The labeled proteins were added to the column and spun down. This step was repeated with the flow-through and final fusion proteins concentrations were measured by BCA.

WT and Batf3 KO mice were injected intradermally in the footpad with a mix of 50 μg of CpG and 6 μg of Alexa 488-labeled Xcl1-(OVA SLP)-Fc or Xcl1-Fc fusion proteins. Inguinal LNs were harvested 16 h post injection for measurement of uptake in different cell populations.

OVA SLP was solubilized with 10% sterile DMSO and 90% sterile PBS. The OVA SLP amino acid sequence is KISQAVHAAHAEINEAGRESIINFEKLTEWT, which includes the MHC class I-restricted epitope (in bold) and the MHC class II-restricted epitope (in italic).

Vaccine formulations were prepared sterile, immediately before injections. Mice were immunized with a volume of 30 μL intradermally in the hind paw, on the ipsilateral side of the tumors.

Mice were engrafted subcutaneously in the left flank either with 1 x 10⁶ EG7 or 2 x 10⁵ B16.OVA cells, or 1 x 10⁵ B16.WT. Tumor volumes were monitored every 2 days and were calculated using the following formula: (length × width × thickness)/2.

Tumor Digestion: Tumors were harvested and digested using the tumor dissociation kit from Miltenyi Biotec (cat number 130-096-730), according to manufacturer's instructions. Cells were then stained for flow cytometry.

Mice received equimolar amounts of Xcl1 and OVA SLP antigen injected intra-dermally in the footpad. Doses were the following: 20 μg of Xcl1-(OVA SLP)-Fc; or 17.6 μg of Xcl1-Fc + 1.3 μg of free OVA SLP; or 1.3 μg of free OVA SLP + 5.9 μg free Xcl1; or 1.3 μg of free OVA SLP. All mice received 50 μg CpG-B (ODN 1826, U133-L01A; Trilink Biotechnologies).

Tumors were digested as described above. Samples were then diluted in 7 mL of complete DMEM and added to 5 mL of Lymphoprep (cat number 1114547, Axis-Shield), followed by a centrifugation of 1,800 rpm for 20 min. Cells at the interphase were collected, washed once, and plated in a 96-well plate for in vitro peptide restimulation.

In vitro peptide restimulation and Intracellular Cytokine Staining: TILs were incubated at 37°C for 1 h with 10 μM SIINFEKL and anti-mouse CD107a (LAMP1) antibody-FITC was also added (1/100) to wells. After 1 h, 1 μg/mL GolgiPlug and GolgiStop (BD biosciences) were added to the wells and TILs were then incubated for a further 4 h at 37°C before intracellular cytokine staining. Cells were permeabilized and stained using the Cytofix/Cytoperm kit (BD Biosciences), according to manufacturer's instructions and stained for intracellular IFNγ and TNFα.

Calculation of the CD8/Tregs ratio: TILs were counted under the microscope before surface/intracellular staining and FACS acquisition. CD8/Treg ratio were calculated using the FACS percentages of tetramer⁺ CTLs and CD4⁺ CD25⁺ FoxP3⁺, and total TIL numbers.

Blood and spleen samples were treated with Red Blood Cell Lysis Solution (Qiagen) for 15 min at 37°C and 3 min at room temperature, respectively, before staining. LIVE/DEAD Aqua fluorescent stain (Invitrogen) was used to discriminate between live and dead cells. For tetramer staining, samples were incubated with phycoerythrin (PE)-conjugated SIINFEKL-H-2k^b multimers (TC Metrix, Switzerland) for 35 min at room temperature. Samples were washed and incubated on ice for 30 min with CD8α-PerCp Cy5.5 (clone 53.6.7–eBioscience), CD3–PE Cy7 (clone 145.2C11–eBioscience), CD4–FITC (clone GK1.5–produced in house, Ludwig Cancer Research). For in vitro binding and chemotaxis assays the following antibodies were used: IgG1–PE (clone A85-1–BD biosciences), B220–Pacific blue (clone RA3-6B2 - LICR), CD8a–PerCp Cy5.5 (clone 53.6.7–eBioscience), CD3–PE Cy7 (clone 145.2C11–eBioscience), CD11c–eFLuor 660 (clone N4/18–eBioscience), CD11b–Alexa700 (clone M1/70–eBioscience), CD103–PE. Data were acquired on a LSRII or LSRII (SORP) and FACS analyses were done with Flow Jo software.

Statistical analyses were performed using GraphPad Prism 7 software (GraphPad Software, La Jolla, CA). Normally distributed data were compared using one-way ANOVA or two-way ANOVA (Figures 3A,B, 5A). Multiple comparisons were corrected using Tukey tests. Normality was tested with a Shapiro-Wilk test. On the graphs, data represent mean ± SE (^*p < 0.05; ^**p < 0.01; ^***p < 0.001; ^****p < 0.0001).

With the aim to optimize synthetic long peptide (SLP) vaccines by targeting the antigen to Xcr1⁺ cross-presenting DCs, a recombinant fusion protein was produced with the ovalbumin (OVA) SLP antigen fused to the Xcl1 chemokine, followed by the murine IgG1 Fc for stability, dimerization and purification purposes (Supplementary Figure 1). We opted for an Fc part harboring the Asp to Ala mutation at amino acid position 265, which prevents its binding to Fc receptors (24). A recombinant protein lacking the OVA SLP antigen (Xcl1-Fc) was also produced to evaluate the potency of Xcl1-mediated antigen targeting (Figure 1A). The fusion proteins were tested for their capacity to bind to CD11c⁺-microbeads purified CD8α⁺ DCs from spleen (Figure 1B). CD11c⁺-enriched DCs from naïve WT and Batf3^−/− mice were incubated with the Xcl1-(OVA SLP)-Fc fusion proteins at 37°C, and specific binding was detected with a fluorescently-labeled anti-IgG1-Fc antibody. Significant binding of Xcl1 fusion proteins was seen in WT mice, when gating on CD11c⁺ CD8α⁺DCs, while some heterogenous non-specific binding was observed on the remaining CD8α⁺ cells from Batf3^−/− mice, which are deficient in Xcr1⁺ DCs (25) (Figure 1C). Similarly, the Xcl1 fusion proteins did not bind to CD8α negative WT and Batf3 KO CD11c⁺ DCs (Figure 1C), supporting the binding specificity to CD11c⁺ CD8α⁺ DCs, 80% of which express Xcr1. To test whether the Xcl1-(OVA SLP)-Fc fusion protein was capable of inducing chemotaxis of Xcr1⁺ DCs, trans-well migration experiments were performed with 1 x 10⁶ CD11c⁺ enriched DCs in the upper chamber and medium containing 25 nM of Xcl1 fusion proteins or commercial Xcl1 in the bottom well. After a 2-h incubation at 37°C, analysis of the bottom well-showed that Xcr1⁺ DCs had migrated between 2 and 4-fold more than Xcr1⁻ DCs in all wells containing Xcl1 fusion proteins or free Xcl1 (Figure 1D). Overall, these data demonstrated that the Xcl1-(OVA SLP)-Fc and Xcl1-Fc proteins induced chemotaxis to a similar extent as the native chemokine Xcl1 (Figure 1D).

To investigate in vivo which population of DCs will preferentially bind the Xcl1-(OVA SLP)-Fc fusion proteins, Xcl1-Fc and Xcl1-(OVA SLP)-Fc were fluorescently-labeled with Alexa 488 and injected intradermally into WT or Batf3^−/− mice. Skin draining LNs were harvested 16 h post immunization and analyzed for the presence of the fusion protein in different subsets of CD11c⁺ DCs (Figure 2A). In WT mice injected with 6 μg of labeled Xcl1-(OVA SLP)-Fc, about 10% of CD8α⁺ LN-resident were Alexa 488 positive, compared to only 2% in Batf3^−/− mice (Figure 2B). Increased uptake of Alexa 488-labeled Xcl1-Fc by WT CD8α⁺ was also observed, as shown by 18% compared to 4.7% in the same DC population in Batf3^−/− mice. With regards to CD103⁺ DCs, there was a tendency for increased uptake of the fusion proteins by WT mice, although not significant due to a large dispersion. Importantly, B cells, which are negative for Xcr1 expression, did not bind the Xcl1 fusion proteins, while < 5% of phagocytic CD11b⁺ DCs, also negative for Xcr1, became Alexa 488 positive for the Xcl1 fusion proteins both in WT and Batf3^−/−, indicating a non-specific uptake (Figure 2C). Altogether, these results suggest that the Xcl1-(OVA SLP)-Fc fusion proteins were preferentially and specifically taken up by the Xcr1⁺ expressing CD8α⁺. Representative profiles of ex vivo Alexa 488⁺-labeled cells are shown in Supplementary Figure 3.

Given that cancer vaccines are ultimately evaluated for their capacity to protect against tumors, the Xcl1 fusion proteins were tested in therapeutic settings against the OVA-expressing EL-4 lymphoma model (EG7). Gender and age-matched C57BL/6 mice were engrafted subcutaneously on day 0 with 1 x 10⁶ EG7 cells (Figure 3A). On day 7, when tumors were established and measurable, mice received an adoptive cell transfer of 10⁵ OT-I cells, followed on day 8 by intradermal vaccination with the Xcl1 fusion proteins or with free OVA SLP +/- Xcl1. Except for the untreated group, all mice received 50 μg of CpG-ODN. In both cohorts vaccinated with the Xcl1-(OVA SLP)-Fc fusion proteins, all tumors started to shrink 5 days post immunization. In contrast, in mice receiving free OVA SLP + free Xcl1, tumor volumes started to decrease only by day 15 but did not disappear, while in mice receiving only the OVA SLP and CpG, only a delay in tumor growth was obtained but no transient decrease of tumor volumes (Figure 3A).

In view of the potent antitumor activity of Xcl1 fusion proteins observed in the EG7 tumor model, we assessed the tumor protective immunity of the Xcl1-mediated tumor vaccine in the less immunogenic B16-OVA melanoma tumor model. Mice were grafted on day 0 with 2 x 10⁵ B16.OVA cells and on day 7, when all tumors were reaching an average volume of 30 mm³, mice received an adoptive cell transfer of 10⁵ naïve OT-I cells, followed on day 8 by the intradermal vaccinations as described for the EG7 challenge (Figure 3A). A significant tumor growth delay was obtained in cohorts vaccinated with Xcl1-(OVA SLP)-Fc and OVA SLP + Xcl1-Fc fusion proteins, as compared to mice not receiving Xcl1 (OVA SLP and CpG only), while only a tendency to a higher delay was observed against the OVA SLP + free Xcl1 cohort (Figure 3B). To assess a non-specific adjuvant effect of the fusion proteins due to potential traces of endotoxin, two groups were vaccinated with the Xcl1-Fc and Xcl1-(OVA SLP)-Fc fusion proteins without CpG. However, both groups of mice showed fast tumor growth (Figure 3B), confirming the adjuvant effect of Xcl1 fusion proteins. As seen in the blood on day 7 post-vaccination in both EG7 and B16.OVA tumor challenge experiments, the vaccination with Xcl1-(OVA SLP)-Fc fusion proteins plus CpG led to similar expansions of OVA-specific CTLs, which was best with Xcl1-(OVA SLP)-Fc, when compared to any other cohort, likely resulting from the co-delivery of the antigen to cross-presenting DCs via its fusion to Xcl1 (Figures 3C,D). Combined immunization with the mixture of the fusion Xcl1-Fc protein and the free OVA SLP + CpG still resulted in a significantly better CTL expansion than in the group receiving free Xcl1 mixed with the OVA SLP + CpG, which only showed a trend for higher OVA-specific CTLs as compared to only OVA SLP + CpG.

In order to dissect the mechanisms by which therapeutic vaccinations using Xcl1 fusion proteins showed better tumor control, B16.OVA tumors from mice immunized as described in Figure 3B, were harvested 10 days post vaccination in order to quantify TILs and characterize their functionality. Frequencies of OVA-specific CD8⁺ T cells in the spleen (Figure 4A) and in the tumors (Figure 4B, left panel) were higher in the cohorts of mice vaccinated with Xcl1 fusion protein as compared to the other cohorts. When normalized by the tumor volume, mice vaccinated with the Xcl1 fusion proteins also showed higher numbers of OVA-specific CD8⁺ T cells, as compared to cohorts vaccinated with free OVA SLP + CpG, with or without free Xcl1 (Figure 4B right panel). Upon in vitro restimulation of tumor-infiltrating lymphocytes (TILs) with SIINFEKL as illustrated in Figure 4C, we found that cohorts vaccinated with Xcl1 fusion proteins showed higher frequencies of IFNγ⁺ TILs than the other cohorts (Figure 4D). Furthermore, increased frequencies of CD8⁺ TILs expressing the lysosomal marker CD107a were also observed (Figure 4E), associated with higher CD107a mean fluorescence intensity (data not shown), indicative of increased degranulation capacity. Altogether, these results suggest not only a higher frequency but also a higher functionality of CTLs within tumors of mice vaccinated with Xcl1-OVA SLP-Fc or Xcl1-Fc + free OVA SLP.

To be closer to a clinical situation, we wanted to assess the tumor protection capacity of the Xcl1 recombinant proteins in therapeutic vaccinations without OT-1 adoptive cell transfer. To this aim, C57BL/6 mice were grafted s.c. with 2 x 10⁵ B16.OVA melanoma cells as described in Figure 3. Mice were vaccinated 3 days later, when tumors were all visible in the flank of the mice. As in the previous experiment involving OT-1 T cell transfer, mice vaccinated with Xcl1-(OVA SLP)-Fc fusion protein showed better control of B16.OVA tumor growth, compared to other cohorts (Figure 5A). Mice were bled 7 days after vaccination and the percentages of OVA-specific CD8⁺ T cells followed the same pattern as seen upon OT-1 cell transfer, with the highest percentages in the Xcl1-(OVA SLP)-Fc and Xcl1-Fc + OVA SLP-immunized mice (Supplementary Figure 2). Strikingly, when comparing tumor growth kinetic with or without OT-1 T cell transfer (Figures 3A, 5A), the tumor control was quite similar, despite a 10-fold lower frequency of endogenous OVA-specific T cells, as seen in the blood on day 7 post vaccination (Supplementary Figure 2). Moreover, when analyzing tumors 10 days post vaccination, we observed that the frequency of OVA-specific CTLs infiltrating the tumors of Xcl1-(OVA SLP)-Fc- and Xcl1-Fc + OVA SLP-immunized mice was only 2–3 fold lower in the absence of OT-1 cell transfer (Figure 5B), which confirmed their efficient homing to the tumor, as compared to mice vaccinated with free OVA SLP + free Xcl1. In addition, these settings also revealed that the ratio between antigen-specific CD8⁺ T cells and Tregs inside the tumor mass was 4-fold higher in Xcl1 fusion proteins-vaccinated cohorts when compared to mice vaccinated with free OVA SLP with or without free Xcl1 (Figure 5C). Representative profiles of the gating strategy for identifying T regs and OVA-specific CTLs are shown in Supplementary Figure 4.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.