Cross-talk between iNKT cells and CD8 T cells in the spleen requires the IL-4/CCL17 axis for the generation of short-lived effector cells

To ensure efficient codelivery of the antigen (ovalbumin, OVA) and the iNKT cell agonist (α-Galcer), we decided to coencapsulate these 2 compounds within polylactic-co-glycolic acid (PLGA) nanovaccines. With this approach, we could work with minimal amounts of antigen and adjuvant and we have demonstrated that vaccination greatly relies on the codelivery, suggesting that efficient iNKT help requires antigen presentation to both CD8⁺ T cells and iNKT cells from the same APC (17). Importantly, we have previously shown that the vaccination efficiency depends solely on the help from iNKT cells and not from CD4⁺ T cells to generate potent OVA-specific CD8⁺ T cells (17). As very limited information is available on how 200-nm-sized PLGA-based nanovaccines (carrying both antigen and adjuvant) enter the spleen and which cells are the primary target, we first examined the capture of fluorescently labeled PLGA nanovaccines in the spleen at early (2 and 6 h) and late time points (24 h) following intravenous injection. The capture was rapid and transient, as we were able to detect nanovaccines in the spleen within 2 h (Fig. 1 A and B), whereas at 24 h, fluorescent nanovaccines were hardly visible (Fig. 1 A and B). This was also confirmed by flow cytometry (SI Appendix, Fig. S1); however, with this technique, CD169⁺ MM could not be assessed for the quantification. As expected, CD68⁺ macrophages and CD11c⁺ DCs formed the main subsets involved in the nanovaccine uptake (∼70% and 30%, respectively, at 6 h) (Fig. 1 C and D). Interestingly, nanovaccines were excluded from the WP, but accumulated at the MZ (Fig. 1A). In accordance, at 6 h about 50% of all nanovaccines detected in the spleen were taken up by CD169⁺ macrophages, followed by CLEC9a⁺ DCs (cDC1) and SIGNR1⁺ macrophages (Fig. 1 C and D). Importantly, the nature of the adjuvant encapsulated in the PLGA nanovaccine did not affect capture by distinct APC subsets, since nanovaccines containing TLR-L (PolyIC+R-848) instead of α-Galcer exhibited the same uptake pattern (Fig. 1D). Thus, nanovaccines are captured by both DCs and macrophages located in the MZ during the first hours after intravenous administration.

We next evaluated whether the iNKT cell adjuvant α-Galcer could induce the production of specific sets of chemokines when compared to more common adjuvants such as TLR-L. To this end, mice were vaccinated and 6 h later different cell subsets were sorted by flow cytometry (CD69⁺ and CD69⁻ OVA-specific CD8⁺ T cells [OT-I] and cDC subsets, XCR1⁺ DC [cDC1] and CD11b⁺ DC [cDC2]) (SI Appendix, Fig. S2). Subsequently, mRNA expression of multiple cytokines, chemokines, and their receptors was measured on sorted populations. Exploiting microarrays, we assessed the expression profile of chemokines and cytokines in OT-I CD8⁺ T cells, based on their activation state (CD69 expression) and the nature of adjuvants carried by the nanovaccine. Performing a nonsupervised clustering, we observed that OT-I CD8⁺ T cells were clustered together, and depended more on the type of adjuvant used than on their activation status (Fig. 2A). This was also true for DCs, whose cytokine/chemokine profiles demonstrated a clustering based on the type of adjuvant rather than on the cDC subset (Fig. 2B). A closer look at the data showed that different chemokine pathways were triggered by the different types of adjuvants (SI Appendix, Table S1). We thus decided to explore this phenomenon in more detail by performing qRT-PCR on the different cell subtypes. CCR4 ligands (CCL17 and CCL22) and CXCR3 ligands (CXCL9 and CXCL10) were specifically induced by α-Galcer nanovaccine (Fig. 2 C and D). In particular, the chemokines CCL17 and CXCL9 were up-regulated by α-Galcer in OT-I T cells and cDCs. In sharp contrast, CCL3 and CCL4 expression, both CCR5 ligands, were specifically induced in activated OT-I T cells of mice that received TLR-L–carrying nanovaccines (Fig. 2 C and D). Finally, IL-12 production, which is important for the initiation of cytotoxic responses, could be detected in XCR1⁺ DC only after administration of nanovaccine containing α-Galcer but not with TLR-L (Fig. 2E). Altogether, these results suggest that iNKT cell help through α-Galcer stimulation has a major impact on splenic chemokine networks by specific induction of CCL17 and CXCL9 expression in multiple immune cell types.

Since we found in various cell types that iNKT cells specifically induce the expression of CCL17 and CXCL9 at mRNA levels, we next sought their protein level distribution within the tissue by confocal microscopy. We confirmed the induction of CXCL9 protein expression upon α-Galcer administration, which is increased over time (Fig. 3 A and B). CCL17 protein expression was also induced 6 h after α-Galcer nanovaccine injection, although its level remained stable during 24 h (Fig. 3 D and E). While expression of both chemokines was restricted to the MZ and RP at early stages (6 h) where nanovaccines were initially detected, they were also found in the WP at later stages (24 h). We showed by flow cytometry that both cDC1 and cDC2 produce these chemokines (SI Appendix, Fig. S3 A and B), confirming our qPCR results. While CXCL9 appeared to be expressed by multiple cell types, CCL17 expression was detected on fewer cells (Fig. 3 A and D), mainly restricted to cDC population (SI Appendix, Fig. S3 C and D). In conclusion, CXCL9 and CCL17 expression is highly dynamic. Importantly, at the early stages, these molecules are produced in the MZ and RP and only start to be expressed in the WP at later stages. This may have important consequences for the migratory behavior of the cells.

Following the cues of T cell-attracting chemokines, we assessed whether T cells were following a similar path. The localization of antigen-specific OT-I CD8⁺ T cells was tracked over time by confocal microscopy. As expected, OT-I T cell behavior was also highly dynamic early after nanovaccine administration in accordance with the chemokine profiles (Fig. 4A). Interestingly, within the first 6 h, almost half of OT-I T cells accumulated at the MZ and in the RP where CXCL9 and CCL17 were detected exclusively (Figs. 3 A–D and 4B). At this stage, OT-I T cells started to get activated, as evidenced by CD69 expression (Fig. 4 C and D). In addition, we evaluated the distance between recruited OT-I T cells and DCs or CD169⁺ macrophages, and we found that they were closer to CD11c⁺ DC and CLEC9a⁺ DC than to CD169⁺ macrophages (SI Appendix, Fig. S4A). This suggests that CD8⁺ T cells were recruited toward DCs rather than to CD169⁺ macrophages during this time frame. Later, from 12 h on, when chemokines became expressed in the WP (Fig. 4 A, B, and D), OT-I T cells were redirected and during this second phase were mainly found in the WP forming stable T–T clusters together with CD11c⁺ DC (Fig. 4 E and F) and CLEc9a⁺ DC but not with CD169⁺ macrophages (SI Appendix, Fig. S4B). At this time, they also started producing IFN-γ and CD69 expression reached a level of ∼90% at 24 h (Fig. 4 C–E). Finally, we questioned the origin of OT-I T cells accumulating outside the WP at 6 h, which can come either from the WP or from the RP. To do so, we compared the location of OT-I T cells that have been injected 1 d before the vaccination or 3 h after (referred to as early migrating OT-I), since it has been shown that 3 h was enough for intravenously injected T cells to home to the WP (18). To accurately quantify this, we compared the ratio of “OT-I/early migrating OT-I” in the WP between control and vaccinated mice. As depicted in SI Appendix, Fig. S4C, we found that the main origin appears to be from the RP, suggesting that T cell trafficking is transiently affected upon nanovaccine administration.

To substantiate these findings, we next studied the migratory behavior of antigen-specific CD8⁺ T cells within various splenic compartments. Since intravital microscopy for the spleen is extremely challenging (19), we opted for an explanted organ approach using perfused thick sections of spleen for live imaging. During early stages after vaccine delivery (2 to 6 h), we observed that OT-I T cells kept their normal high-speed motility of around 7 μm/min in the WP as at the steady state (Fig. 5 A and B and Movie S1). In the MZ and the RP, OT-I T cells exhibited a somewhat slower speed with a mean velocity of 5 μm/min (Fig. 5 C and D and Movie S2). This slowing could result from repetitive short encounters with APCs. This notion was supported by the finding that in the absence of OVA antigen or with polyclonal CD8⁺ T cells, the velocity was slightly but significantly higher in those regions during this time frame (Fig. 5 C and D). During the second phase (12 to 24 h), when T cells were redirected to the WP, OVA-primed T cells significantly slowed down their migratory behavior (Fig. 5 A and B), reflecting the formation of stable conjugates together with DCs (Fig. 4 E and F and Movie S3). Altogether, these results demonstrate that antigen-specific CD8⁺ T cells exhibit a biphasic behavior, with a first transient accumulation at the MZ and the RP early after nanovaccine administration, where they interact shortly with DCs, and at later stages with the recruitment of CD8⁺ T cells in the WP, with long-lasting contacts involving multicellular clusters with DC.

Since our results point out that both CCL17 and CXCL9 are important chemokines in our vaccination strategy, we decided to evaluate the expression of their respective receptors, CCR4 and CXCR3, on OT-I T cells.

At the steady state, a fraction of naive OT-I T cells already harbors CXCR3 at the cell surface, thus rendering these cells responsive to CXCL9 at very early stages. In contrast, CCR4 could not be detected at the cell surface of naive cells and became only up-regulated after vaccination (Fig. 6A). In order to reveal which factors might induce its expression on the cell surface over time, we cultured in vitro purified OT-I T cells in the presence of IL-4 combined or not with OT-I–specific peptide (SIINFEKL) to trigger T cell activation and monitored CCR4 expression by flow cytometry. We found that only the combination of IL-4 and SIINFEKL induced a strong expression of CCR4, while cells exposed to either of them alone only induced a mild expression (SI Appendix, Fig. S5A). CCR4 expression was rapidly induced since it could already be detected from 6 h on, although still higher expression levels were seen at 24 h. Coming back to in vivo vaccination setting, we were able to detect surface expression of CXCR3 on OT-I T cells at early stages of vaccination (6 h). However, the levels of mRNA expression and protein were lower when compared to the control (Fig. 6A and SI Appendix, Fig. S5B). Nonetheless, the vast majority of detected CXCR3⁺ OT-I T cells expressed CD69, suggesting an important role of this chemokine receptor to recruit antigen-specific T cells in the early stages of activation. At 6 h, the expression of CCR4 was up-regulated at the mRNA level but was not detectable at the cell surface (Fig. 6A and SI Appendix, Fig. S3B). Perhaps engagement of the receptor by its ligand induces its internalization or prevents its recognition by the antibody when the epitope is shielded. At 24 h, the proportion of OT-I T cells expressing CXCR3 strongly increased and CCR4 expression was detected on a fraction of activated CD69⁺ OT-I T cells (Fig. 6A). We then evaluated the contribution of these chemokine receptors in vivo for early T cell activation and recruitment in the WP. Interestingly, we found that blocking each of them impacted early T cell activation monitored by CD69 expression 6 h after vaccination (Fig. 6B). Furthermore, we saw that the recruitment of OT-I T cells in the WP was negatively impacted by CXCR3 or CCR4 blockade, as depicted in Fig. 6C. Altogether, these results show that the expression of CXCR3 and CCR4 on antigen-specific T cells is triggered upon vaccination and participate in their activation and recruitment.

Since CD8⁺ T cells were rapidly recruited to the MZ and the RP after nanovaccine administration, we hypothesized that they could encounter already activated iNKT cells in these areas during this first phase. Therefore, we first examined whether iNKT cells were activated within the first 6 h upon nanovaccine administration (SI Appendix, Fig. S6A). As expected, we found that most of the iNKT cells were indeed activated according to their CD69 expression.

We then attempted to directly visualize interactions between OT-I T cells and iNKT cells by confocal microscopy. Adoptive transfer of highly purified and fluorescently labeled iNKT cells was not an appropriate method since it did not result in sufficient numbers in the spleen for obtaining solid data. A better approach was to stain endogenous iNKT cells with a CD1d–α-Galcer dextramer and to image OT-I T cell/iNKT cell interactions in fixed tissue sections. As depicted in SI Appendix, Fig. S6B, close interactions between OT-I T cells and iNKT cells could be observed in the MZ and in the RP. By quantifying the distance between OT-I and iNKT cells in these regions, we could demonstrate that these close interactions only occurred from 6 h after vaccination (SI Appendix, Fig. S6C). Thus, while OT-I T cells accumulated at the MZ and in the RP, they could form concomitant interactions together with iNKT cells.

Such an approach was unfortunately limited by the rapid down-regulation of TCR expression on iNKT cell surface triggered by their activation, rendering the quantification difficult after 6 h. To circumvent this drawback and to further study these interactions over prolonged periods of time, we exploited CXCR6-GFP mice, which have been already used in intravital microscopy studies to track NKT cells in the liver (20). As depicted in Fig. 7A, in these mice, CXCR6-GFP^bright cells represent a homogenous population in the spleen and the vast majority of them are NKT cells (based on coexpression of CD3 and NK1.1). Based on CD1d–α-Galcer dextramer binding, 62% of this population can be designated as type I NKT cells or iNKT cells (CD1d-restricted NKT cells recognizing α-Galcer) and the remainder of the cells are most likely type II NKT cells, which do not bear a specific marker. Furthermore, upon vaccination, the CXCR6-GFP^bright cell population was not increasing and remained representing NKT cells (SI Appendix, Fig. S7A). Assessment of NKT cell distribution in these mice revealed that their localization followed the same biphasic dynamics as OT-I T cells (Figs. 4B and 7 B and C). During the first phase (6 h), they were localized to the MZ and RP where they formed NKT–NKT clusters (Fig. 7D), whereas during the second phase (24 h), most of them were found in the WP where NKT-NKT clusters could still be observed. By evaluating the interactions between NKT cells and OT-I T cells, we were able to reveal close contacts (less than 3 µm) between NKT cells and OT-I T cells together with DCs (Fig. 7 E and F). Indeed, while ∼20% of OT-I cells were found in close contact with NKT cells in the MZ and the RP in the first phase (6 h), almost half of OT-I cells were found in concomitant interactions in the WP in the second phase (24 h) (Fig. 7E).

To establish the identity of CXCR6-GFP^bright cells, we confirmed that they were NOT CD8⁺ T cells by immunostaining, as illustrated in the SI Appendix, Fig. S7B. Of course, we cannot guarantee that the observed contacts are all with-type I NKT cells (iNKT cells), since they represent 60% of the CXCR6-GFP^bright cell population (forming the main population), but we assume that in the majority of the quantified contacts, iNKT cells are involved. By live imaging, we quantified the duration of these contacts and found that these interactions were short and independent of the presence of the OVA antigen at the early stages (Fig. 7G and Movie S4). However, at the late stages, we observed long-lasting interactions with about half of the contacts that lasted at least 30 min (duration of the entire movie shown in Movie S5). To exclude that the observed interactions could only be attributed by the high density of both OT-I and NKT cells in the WP at the late stages, we randomly simulated the position of NKT cell in these regions. As shown in Fig. 7H, we found that the distance between OT-I and NKT cells was closer in reality than when distributed randomly, supporting unambiguously a true connection between NKT and OT-I T cells.

Finally, we addressed the role of CXCR3 and CCR4 in the recruitment of NKT cells in the WP at the late stages and found, surprisingly, that CXCR3 and CCR4 have a differential role in this process (Fig. 7I). Indeed, CXCR3 plays a positive role in this process while, in contrast to T cells, CCR4 negatively impacts this migration. Based on these findings, we demonstrate that NKT cells are first recruited in the MZ and in the RP, where they get activated and can interact shortly with OT-I T cells. In the second phase, they follow the behavior of OT-I T cells by being recruited toward the WP, where they form concomitant long-lasting interactions with DCs.

We next addressed the functional impact of CXCR3 and CCR4 on the generation of CD8⁺ T cell memory. In particular, we focused on CCR4 since the role of this chemokine receptor in this process was not really explored.

First, we aimed to confirm in vivo the link between IL-4 and the CCR4 pathway, as we previously showed in vitro in SI Appendix, Fig. S5A. As expected, we found that IL-4 was only detected in the sera of animals administrated with nanovaccines containing an iNKT cell agonist (Fig. 8A). Maximal production was found in the early stages (6 h) when OT-I T cells accumulate outside the WP. Interestingly, we could detect IL-4 signaling in OT-I T cells by pSTAT6 staining only during this time frame and not in the late stages (Fig. 8B). We then evaluated the impact of IL-4 blockade in vivo on the expression of CCR4 on OT-I T cells and CCL17 production in the spleen. We found that IL-4 blockade strongly reduced CCR4 levels on OT-I T cells (Fig. 8C) and was sufficient to abolish the production of CCL17 in the spleen (Fig. 8D), thus confirming the key role of IL-4 in the induction of the CCR4 pathway.

We then studied the consequence of either CXCR3 blockade or IL-4/CCL17 blockade at the time of the vaccination on the generation of memory precursor cells (MPEC) or SLEC 10 d postnanovaccines administration. Interestingly, we found that blocking CXCR3 drastically reduced the numbers of antigen-specific T cells at day 10 postvaccination (Fig. 8E). This can mainly be explained by the strong reduction of the generation of MPEC in these conditions (Fig. 8F). As a consequence, a higher proportion of SLEC was found. Conversely, blockade of either CCL17 or IL-4 did not negatively impact the numbers of antigen-specific T cells at day 10, with even a slight tendency to increase (Fig. 8G). Importantly, the blockade of each of these molecules reduced the generation of SLEC and increased the proportion of MPEC in a completely opposite fashion to CXCR3 (Fig. 8H). Thus, CXCR3 and CCR4 have clear differential roles in the generation of MPEC or SLEC, respectively.

Since the IL-4/CCL17/CCR4 axis is crucial for the generation of SLEC and IL-4 is only produced during the early stages (phase 1), we hypothesized that early cross-talk between iNKT and CD8⁺ T cells might be important for the generation of SLEC.

To test that, we unsynchronized the delivery of antigen and the iNKT cell agonist by administrating nanovaccines (nanoparticles, NP) containing OVA at the same time, 10 h after (postphase 1) or 48 h after (postphases 1+2) the injection of NP containing α-Galcer, as illustrated in Fig. 9A. We first evaluated the expansion of antigen-specific T cells 10 d after antigen administration and we found that both phases are crucial in this process (Fig. 9B). Then, we studied as previously the differentiation of antigen-specific T cells into MPEC or SLEC at day 10 (Fig. 9C). Interestingly, we found that both phases are important for the generation of MPEC when we looked at the relative numbers of MPEC OT-I T cells (Fig. 9D). More importantly, we saw that only the absence of the phase 1 is enough to nearly abolish the generation of SLEC (Fig. 9E) and that phase 2 did not give any additive effects. Thus, as predicted, the early cross-talk (phase 1) between iNKT and CD8 T cells is crucial for the efficiency of the nanovaccine and the generation of SLECs.

To summarize, we propose a model in which iNKT–CD8 T cell cross-talk occurs in 2 spatiotemporal phases and involves simultaneous interactions between 3 cell types (SI Appendix, Fig. S8). The first phase (2 to 6 h postvaccination) occurs in the MZ and the RP where recruited OT-I T cells interact shortly with activated iNKT cells and DC. During the second phase (12 to 24 h), OT-I T cells, iNKT cells, and DC establish long-lasting contacts forming a “ménage à trois” in the WP. In this process, CXCR3 and CCR4 play a differential role. The IFN-γ/CXCR3 pathway will be involved in the generation memory CD8 T cells, while the IL-4/CCR4 pathway will regulate the generation of SLECs. We believe that our study highlights the importance of iNKT cells as helper cells to orchestrate the complex dynamic process of cross-priming in the spleen and its importance for proper memory T cell generation.

All animal work was done in accordance with Institutional Animal Care and Use Guidelines of the Central Animal Laboratory (Nijmegen, The Netherlands). All mice were housed in specific pathogen-free conditions before use. Wild-type C57/BL6J mice were from the Charles River, OT-I yeti or Rag1^−/− OT-I mice were bred in house. CXCR6-GFP mice were kindly provided by the Rachel Golub laboratory, Pasteur Institute, Paris, France, and were maintained as heterozygous at the animal facility of the Cochin Institute Paris, France.

The following antibodies were used for flow cytometry: anti–CD8α (53-6.7), anti-CD44 (IM7), anti-CD45.1 (A20), anti-CD45.2 (104), anti-CD62L (MEL-14), anti-CD69 (H1.2F3), anti-CD127 (A7R34), anti–KLGR-1 (MAFA), anti-mPDCA1 (129C1), anti-V_α2 (B20.1), anti-NK1.1 (PK136), anti-B220 (RA3-6B2), anti-CD11b (M1/70), anti-CD11c (N418), anti-F4/80 (BM8), anti–MHC-II (M5/114.15.2), anti-CCR4 (2G12), anti-CXCR3 (CXCR3-173), anti-XCR1(ZET), anti-CD3 (145-2C11), anti-CD5 (53-7.3), and anti-TCRβ (H57-597), all from Biolegend; anti-Vβ5.1/5.2 (MR9-4) from eBioscience.

The following antibodies were used for microscopy: anti-CD169 (3D6.112), anti-CD11c (N418), anti-CD68 (FA-11), and anti-CXCL9 (MIG-2F5.5), all from Biolegend; anti-mCLEC9a (polyclonal sheep IgG) and anti-mCCL17 (polyclonal goat IgG), both from R&D systems; anti-CD3 (polyclonal rabbit) from Abcam; anti-SIGNR1 from AbD Serotech.

CD1d-α-GalCer dextramer was obtained from Immudex. Corresponding secondary antibodies were bought either from Life Technogies (donkey anti-goat) or from Jackson Immunoresearch Laboratories [Goat anti-hamster, Fab′ (2) donkey anti-rat, donkey anti-rabbit, donkey anti-sheep].

PLGA (Resomer RG 502 H, lactide/glycolide molar ratio 48:52 to 52:48) was purchased from Boehringer Ingelheim. Solvents for PLGA preparation (dichloromethane) were obtained from Merck. Polyvinyl alcohol (PVA) was obtained from Sigma. R848 was from Enzo Life Sciences, poly I:C from Sigma-Aldrich, and endotoxin-free OVA from Hyglos. α-GalCer was purchased from Funakoshi. DMSO 99.9%, Triton-X100, and Tween 20 was obtained from Sigma. Atto647N was obtained from Atto-tec. RPMI medium 1640 was from Life Technologies. Celltrace violet (CTV) and Celltrace far red (CFR) were purchased from Life Technologies. SIINFEKL peptide was obtained from Anaspec. BSA was bought from Roche. SuperScriptII, RNase OUT (RNase inhibitor), First Strand Buffer, Second Strand Buffer, DNA polymerase I (Escherichia coli), 0.1M DTT, RNase H (E. coli) were obtained from Invitrogen. AMPure XP beads and RNAClean XP beads were obtained from Beckman Coulter.

PLGA nanovaccines were manufactured and characterized as described previously (17).

Spleens were harvested and digested with DNase I (Thermofisher) and Collagenase III (Worthington) at 37 °C for 30 min. Organs were then mechanically digested and passed over 100-µm cell strainers (Corning). Red blood cell lysis was performed with 2 mL of 1× ammonium chloride solution for 5 min at room temperature. Cell suspension was then stained with fluorescently labeled antibody in PBA (PBS 1% BSA 0.01% sodium azide) for 20 min at 4 °C in presence of CD16/CD32 blocking (24G2 BD Pharmingen). For iNKT staining, cells were incubated 10 min with fluorescently labeled CD1d-αGalCer dextramer at room temperature in PBA prior incubation with fluorescently labeled antibodies. Cells were analyzed with FACS verse, FACS LSRII, or FACS Fortessa (BD). Results were analyzed with FlowJo (Tree Star).

OT-I CD8⁺ T cells were isolated from spleen and peripheral LNs by a negative selection kit (CD8α isolation kit mouse Miltenyi) with added biotinylated anti-CD44 antibodies according to Gerner et al. (38). Purity was routinely superior to 95%. OT-I cells were labeled with either CTV (Life Technologies) or CFR (Life Technologies) with a dye concentration of 5 µM following the manufacturer’s procedure. Next, 1 to 2 × 10⁶ dye-labeled OT-I CD8⁺ T cells were adoptively transferred into congenic wild-type mice, followed by intravenous injection of nanovaccine 1 d later with a dose corresponding to 1 µg OVA, 1.5 ng α-GalCer, 215 ng R848, and 105 ng poly I:C. For the blocking experiment, 500 μg anti-CXCR3 blocking antibodies, 300 µg of IL-4 or CCL17 antibodies, or their corresponding isotype (Bioxcell/R&D systems) were injected intraperitoneally 1 d prior to vaccination. Alternatively, 2.5 μg of CCR4 antagonist (CAS 864289-85-0 Cayman) or the vehicle (0,05% DMSO) was injected intraperitoneally 1 d before and at the same time of vaccination.

For the MPEC and SLEC experiments, 1.000 purified OT-I CD8⁺ T cells were intravenously injected into CD45.1 animals. Nanovaccines were administrated intravenously the following day, and 10 d later, mice were killed, and spleens harvested and analyzed by flow cytometry.

For in vivo T cell homing, 500 × 10³ purified CTV labeled OT-I CD8⁺ T cells were intravenously injected into wild-type animals 1 d prior to vaccination. Nanovaccines were administrated intravenously the following day, and 3 h later 500 × 10³ purified CFR-labeled OT-I CD8⁺ T cells (representing “early migrating OT-I cells”) were intravenously injected. Three hours later (6 h postvaccination), mice were killed, and spleens were harvested and fixed for subsequent immunofluorescence staining. The ratio of CTV OT-I/CFROT-I was calculated in the WP on the different images in the control mice and normalized to 1. The ratio of CTV OT-I/CFR OT-I was calculated in the vaccinated mice and compared to the control.

In order to validate the significance of possible “contacts” between NKT cells and OT-I cells in the WP, we developed an R Shiny application using the R package ‘shiny’ version (https://CRAN.R-project.org/package=shiny) (39). The NKT cells and OT-I cells positions and the contour of the WP were extracted manually from different images with Image J (NIH). The R program calculates the mean of the closest distance between the OT-I cells and NKT cells for each OT-I T cell (using nncross: Nearest Neighbors Between Two Patterns from spatstat package). In order to simulate the effect of density to the percentage of cells in possible contact, we ran 20 simulations of random positions of NKT in the same region of interest (WP) and calculated the mean of the closest distance between OT-I and NKT cells for each analyzed picture.

Spleens were fixed with 4% PFA for 2 h on ice. Successive baths of 10%, 20%, and 30% fructose were performed at 4 °C for 48 h. Organs were then embedded in OCT (Sakura), snap-frozen, and stored at −80 °C. Ten- to 20-μm cryosections were performed with a microtome (Microm HM 500 Cryostat) at temperatures between −18 °C and −21 °C. For immunostainings, residual PFA was quenched with PBS 0.1M Glycine for 5 min at room temperature, sections were then stained overnight at 4 °C with primary antibodies in PBA 0.1% Triton-X100. After washing with PBA, sections were then stained in PBA with secondary antibodies 2 h at 4 °C, washed, and then counterstained with DAPI (Sigma) if needed. Sections were finally washed in PBS and then in milliQ and mounted with a coverslip and Prolong Antifade Diamond mounting medium (Life Technologies). Confocal microscopy was performed with Olympus FV1000 or Zeiss LSM880 with 20× or 40× objectives. Pictures were then analyzed with ImageJ. To analyze NP uptake or chemokine expression, a background threshold was applied to quantify the total fluorescence intensities. Concerning the calculation for fluorescence intensities per square micrometer, fluorescence background was subtracted and then pictures were analyzed. Percentage of cells per area was performed on the total of cells in the whole section. Cells with a minimal distance shorter than 3 μm were considered to be in close contact.

Spleens were embedded in a solution of 7.5% low gelling-temperature agarose (type VII-A; Sigma-Aldrich) prepared in PBS. Slices (500 μm) were cut with a vibratome (VT 1000S; Leica) in a bath of ice-cold PBS. Slices were then transferred to 0.4-μm organotypic culture inserts (Millicell; Millipore) in 35-mm Petri dishes containing 1 mL RPMI 1640 without Phenol red. Live vibratome sections were stained for 15 min at 37 °C with antibodies. All antibodies were diluted in RPMI without Phenol red and used at a concentration of 10 μg/mL. Tissue sections were imaged with a DM500B upright microscope equipped with an upright spinning-disk confocal microscope (Leica) in a 37 °C thermostated chamber. For dynamic imaging, tumor slices were secured with a stainless steel slice anchor (Warner Instruments) and perfused at a rate of 0.8 mL/min with a solution of RPMI without Phenol red, bubbled with 95% O₂, and 5% CO₂. Next, 10× or 25× water-immersion objectives were used and 30-min movies of different areas were recorded. Velocity was calculated using the plugin Manual Tracking from ImageJ. For CD1d/α-GalCer staining, thick sections were stained in PBA for 30 min at room temperature, and then 2 h on ice with CD169 and CD11c antibodies. Thick sections were then fixed 20 min at room temperature with 4% PFA, washed with PBS, and then imaged by confocal microscopy.

For in vitro culture, 50 × 10³ purified OT-I CD8⁺ T cells were cultured in a96U-well plate with a complete medium (RPMI 1640 supplemented with 10% FCS, nonessential amino acid [Gibco], l-glutamine [Gibco], anti-anti [Gibco], sodium pyruvate [Gibco], Hepes, and β-mercaptoethanol) at 37 °C for 5 or 24 h. Concentrations of SIINFEKL and IL-4 were, respectively, 1 μg/mL and 10 ng/mL.

For statistical analysis, 1-way ANOVA or Mann–Whitney t test was used with Graphpad; *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.

All data supporting the findings of this study have been deposited in Figshare.