which is itself necessary for the production of IL-12. The relation between tumour growth and IL-4 has been reported for numerous types of cancer expressing IL-4 receptor (IL-4R) including NPC. In fact, Budiani and colleagues reported an increase in IL-4 production both at the tumour level and in the sera of NPC patients compared to controls (Budiani et al., 2011; Shurin et al., 1999). IL-4 plays a critical role in tumour development, being associated with aggressiveness and metastasis potential (Kobayashi et al., 1998), and most recent studies indicate that endogenous IL-4 can favour resistance to apoptosis via IL4-R signalling (Li et al., 2008). Very interestingly, we also pointed out, the ability of NPCSEV-DCs to secrete much more IL-4 than other classical DCs, either mature or tolerogenic. Therefore, altogether, our results clearly indicates that the emerging NPCSEV-DCs belong to the newly described mregDCs subtype.

More recently, in an attempt to understand the failure of phase III clinical trial targeting IDO associated to immune checkpoint blockade, Sadik et al. has described, and largely proved, that interleukin 4 (IL-4)-induced gene 1 (IL4I1), a secreted enzyme belonging to the L-amino-acid oxidase family, was able to promote Aryl-Hydrocarbon Receptor (AHR)-driven cancer immunosuppressive ability. This potency was even described as more effective than for IDO1 or IDO2, hitherto recognized as the main Trp-catabolic enzymes in the TME (Boulland et al., 2007; Sadik et al., 2020). Interestingly NPCSEV-DCs produce IL4I1 and the fact that mDCs produce as much as IL4I1 than tolerogenic DCs, but without suppressive influence, could probably be explained by a restriction of its activity (or the one of its metabolites) in the effector microenvironment, while emphasized in an immunosuppressive one. A point that need to be clarified with further experiment. Thanks to our results, we can assume that the immunosuppressive properties do not only require or rely an increase in IL4I1 secretion, but probably much more via the concomitant action of the immunosuppressive molecules released after NPC derived EVs contact. Nevertheless, it is interesting to note that according to the TCGA dataset covering more than 500 H&N cancer patients, IL4I1 is significantly more than two-fold overexpressed in tumour tissue than in normal ones (Supplementary data 7A). While looking at the most probable source of the enzyme into the TME of H&N cancer patients, one can observe a strong correlation with numbers of immune infiltrating cells, and among them myeloid cells are the most correlated (Supplementary data 7B); confirming our data in NPCDC ex vivo assays. Furthermore, when examining the impact of IL4I1 expression in the survival of H&N cancer patients, we found that, although it does not appear to impact on the overall survival (OS) of patients, there is a significant difference in relapse-free survival (RFS, Supplementary data 7 C.a and C.b). As a result, the high IL4I1 expression is, the more likely patients are to relapse, confirming that IL4I1 could be a good candidate for therapeutic development. It is important to note that the main immune checkpoints described to date (PDI, PDL1, CTLA4) have exactly the same profiles for most cancers. Overall, our data are in agreement with several other recent publications (Molinier-Frenkel et al., 2019; Sadik et al., 2020), and it can be assumed that IL4I1 is a novel immune checkpoint, and an interesting target for the development of new antitumor therapies. Finally, Maier et al., in their last description of mregDCs indicates that, IL4I1 was upregulated among other Th2 markers. Altogether, this information supports that IL-4 upregulation and consequently IL4I1 secretion represent a real hallmark of mregDCs, explaining their detrimental role in cancer progression and resistance (Maier et al., 2020). The last authors suggested that some help in counteracting tumour resistance to treatment in lung cancer patient could come from an IL-4 blockade, and according to our results this adjuvant treatment could surely be of great help to overcome resistance and delay recurrence in non-responding NPC patients.

In NPC context, another particularly interesting candidate is Galectin-9 (Gal9) that is found on NPC SEVs (Keryer-Bibens et al., 2006; Pioche-Durieu et al., 2005) and has various immunosuppressive capacities that greatly impact on the anti-tumour response. Indeed, galectin-9 lead to Th1 cells apoptosis (Zhu et al., 2005), decrease secretion of pro-inflammatory cytokines (IL-17, IL-12 and IFN-γ (Seki et al., 2008), and favor MDSCs or iTreg generation (Seki et al., 2008; Wu et al., 2014; Zhang et al., 2020). More importantly, it was reported that exosomes isolated from the cerebrospinal fluid (CSF) of patients with glioblastoma multiform (GBM) contain galectin-9 that can interact with the TIM3 receptor on DCs. This interaction inhibits antigen recognition, processing and presentation by tumour-infiltrating DCs, resulting in the failure of the cytotoxic T-cell-mediated anti-tumour immune responses (Ming et al., 2020). Studies have demonstrated the importance of the Galectin-9/TIM3 interaction in the regulation of dendritic cell function in cancer. Blocking TIM3 receptor on cDC1 in breast cancer has shown an improved anti-tumour response after chemotherapy by promoting intra-tumour CD8+ T response mediated by CXCL9 expression. Blocking Galectin-9 in this same model showed the same results as anti-TIM3 with an increase of CXCL9 expression confirming the interaction between these two actors in DC modulation effect (de Mingo Pulido et al., 2018). Recently, it has been described in the context of chronic coxsackievirus B3 infection that Gal9 promotes LT CD4 anergy, enhances Treg emergence and IL-10 and IL-4 production (Lv et al., 2011). This result, in line with ours, led us hypothesize exosomale Gal9 could be an interesting candidate to explain our results especially in the context where we had shown that NPC derived SEVs directly influenced Tregs (Mrizak et al., 2015).

TIM3, the main receptor of Gal9, is a transmembrane protein member of the TIM family of immunoregulatory receptors with immunosuppressive functions (Wolf et al., 2020). There are no classical inhibitory signalling motifs in the cytoplasmic tail of TIM3 which contains only five tyrosine conserved between mice and humans. Among those tyrosines, Tyr256 and Tyr263 allow interactions with HLA- B-associated transcript 3 (BAT3) (Rangachari et al., 2012) and the tyrosine kinase FYN (Lee et al., 2011). According to current knowledge, when TIM3 is free of ligand, BAT3 is retain at its cytoplasmic tail and helps in activating signalling of T cell (Clayton et al., 2014). However, upon Gal9 binding, TIM3 oligomerize and both Tyr256 and Tyr263 are phosphorylated, which secondly triggers BAT3 release and unleash immunosuppressive function of TIM3 (van de Weyer et al.,
2006; C. Zhu et al., 2005). Although firstly described as a T cell membrane molecule (Monney et al., 2002), Tim3 was found to be expressed in several myeloid cells such as neutrophils (Anderson et al., 2007; Huang et al., 2020), monocytes/macrophages (Nakayama et al., 2009; Vega-Carrascal et al., 2014; Zhang et al., 2020) and more recently on Dendritic cells (Chiba et al., 2012; de Mingo Pulido et al., 2018), usually at higher levels than observed in T cells. The activation of the Gal9/Tim3 axis on myeloid cells regulate their polarization and turn them into immunoregulatory cells. For example, it has clearly been demonstrated that Gal9 could promote M2 type macrophages by deeply modifying expression patterns of transcription factors with an increase of STAT3 phosphorylation as well as cytokines release with a IL-12/IL-23 axis blockade combine to an increase of IL-10 and TGF-β secretion (R. Lv et al., 2017; Ma et al., 2013). On intratumor CD103+ cDCs, TIM-3 decrease the expression of Cxcl9 and Cxcl10 chemokines, implicating its role in effector T cell fate (de Mingo Pulido et al., 2018). More recently, the same team has described a mechanism by which TIM-3 suppresses DNA sensing through the cGAS-STING pathway through the binding of Gal9 (de Mingo Pulido et al., 2021). This idea was recently further reinforced by the demonstration of the direct role played by Gal9 on the degradation of STING shifting the myeloid compartment through the immunoregulatory part promoting immunosuppressive microenvironment in Gal9 secreting tumour (Zhang et al., 2020). The latter's research were also perfectly correlated with another recent one indicating that a blockade of TIM3 could not only increase antitumor CD8 T cells activation and IFN-γ secretion, but would also favour cDC1 cells and CD8 T cells to colocalize within the tumour, amplifying the chance to mount an appropriate antitumor response both in time and space (Gardner et al., 2022). Moreover, it has been very recently described that BAT3 is a specific and endogenous regulator of tolerogenic DCs phenotype and function able to favour a pro-tumour T cell compartment (reduction of TH1 cells, and increase in Treg cells) (Tang et al., 2022). Finally, in term of metabolic activity, it is now clear that TIM3 and Gal9 act as enhancer of immunoregulatory function through a shift in glycolysis at the expense of OXPHOS. This has been well described for Treg cells (Banerjee et al., 2021) and due to well established knowledge about tolerogenic DCs, one can speculate the same feature for them (Dáňová et al., 2015). Altogether, these studies indicate clearly the major role displayed by the Gal9/TIM3 axis, both in CIS and TRANS, in the emergence of regulatory DCs, notably within the tumour microenvironment. Indeed, one can resume that Gal9 binding to TIM3 on DC reinforce its membrane stability, allows TIM3 to oligomerize in order to deliver its immunoregulatory signal. This signal relies both on the degradation of STING and on the release of BAT3 protective adapter from the cytoplasmic tail of TIM3. These two complementary signals lead to the activation of STA3 and NFκB transcription factors resulting in blocking IL-12 secretion in favour of IL-10 and IL-6 release (Barton, 2006), such as described for tumour cells (Zhang et al., 2018). Because of this dysregulation, Dendritic Cells lose their ability to activate antitumor T cells response and rather facilitate tolerance in T cells via multiple mechanisms already described elsewhere such as T cell anergy, T cell depletion or conversion into FOXP3 regulatory T cells (Iberg et al., 2017).

In the present study, we described the emergence of tolerogenic mregDCs after exposure to Gal9+ NPC derived SEVs. Indeed, all the event described above are representative and perfectly in line with what could happen to DCs during their development in the presence of NPC SEVs in the tumour context, and the consequence in term of regulatory gene overexpression (Supplementary data 6 B and D), metabolism shift, and both cytokine release and T cell anergy is strongly in favour of the role of Gal9/TIM3 axis so. Nevertheless, to prove indisputably the role of Gal9 on this process, we decided to move further and use a specific mAb able to block its capacities as we described with our collaborators (Lhuillier et al., 2018; Sarahjibrin, 2021). Our results indicate that this antibody is able to block T cell death or anergy triggered by recombinant Galectine-9. In the present work, we used this original anti-Gal9 blocking antibody (1g3) that allows us to prove that the immunosuppressive Gal9 could partly be responsible for the change in the fate of DC in its secreted form but overall associated to extracellular vesicles. This indication reinforces our understanding of the immunosuppressive role played by Gal9 itself in NPC and the ability of EVs to convey this property on target cells.

Finally, in parallel to previous work from the team demonstrating that NPC-Exo could recruit Tregs at the tumour site both In vitro and in vivo (Mrizak et al., 2015), we intend to demonstrate that NPCSEVs preferentially attract NPCSEV-DC which could also enhance the suppressive microenvironment present in NPC. Interestingly we observed that tDC do not migrate under any chemotactic gradient as shown in the migration movie of tDC under a C15SEV gradient (Supplementary data 1). This could be explained by the use of vitamin D3 and dexamethasone to generate them. Indeed, another study reports similar results with tDC generated with vitamin D3 or dexamethasone that do not migrate toward a CCL19 gradient and have an under-expression of CCR7, the CCL19 receptor (Adnan et al., 2016; Barragan et al., 2015). It is important to note that glucocorticoids as dexamethasone are able to cause microtubule and F-actin rearrangement in T lymphocytes leading to depolarization of the cells preventing their migration (Müller et al., 2013). Same action was described in mesenchymal stem cell that loss migration behaviour after dexamethasone treatment (Schneider et al., 2015) and could have led to the paralytic state of tDC that we observed.

Given that the presence of conventional DCs can be considered very early in tumour development and given the role of mregDCs in resistance to anti-cancer treatments, it can be assumed that SEVs are one of the main players in this feature. Above immunotherapies, this cellular mechanism could probably explain, at least in part, the fatal resistance observed in too many NPC patients after recurrence of the classical radio-chemo therapeutic approach.

Altogether our data, offers a new point of view about the role of NPC derived SEV on DCs. This study presents for the first time that tumour derived small extracellular vesicles contribute to NPC immune evasion via the emergence of mregDCs through Gal9. This indirect mechanism enhances the lack of proinflammatory cytokines production and decrease capacity to activate effector
2006; C. Zhu et al., 2005). Although firstly described as a T cell membrane molecule (Monney et al., 2002), Tim3 was found to be expressed in several myeloid cells such as neutrophils (Anderson et al., 2007; Huang et al., 2020), monocytes/macrophages (Nakayama et al., 2009; Vega-Carrascal et al., 2014; Zhang et al., 2020) and more recently on Dendritic cells (Chiba et al., 2012; de Mingo Pulido et al., 2018), usually at higher levels than observed in T cells. The activation of the Gal9/Tim3 axis on myeloid cells regulate their polarization and turn them into immunoregulatory cells. For example, it has clearly been demonstrated that Gal9 could promote M2 type macrophages by deeply modifying expression patterns of transcription factors with an increase of STAT3 phosphorylation as well as cytokines release with a IL-12/IL-23 axis blockade combine to an increase of IL-10 and TGF-β secretion (R. Lv et al., 2017; Ma et al., 2013). On intratumor CD103+ cDCs, TIM-3 decrease the expression of Cxcl9 and Cxcl10 chemokines, implicating its role in effector T cell fate (de Mingo Pulido et al., 2018). More recently, the same team has described a mechanism by which TIM-3 suppresses DNA sensing through the cGAS-STING pathway through the binding of Gal9 (de Mingo Pulido et al., 2021). This idea was recently further reinforced by the demonstration of the direct role played by Gal9 on the degradation of STING shifting the myeloid compartment through the immunoregulatory part promoting immunosuppressive microenvironment in Gal9 secreting tumour (Zhang et al., 2020). The latter's research were also perfectly correlated with another recent one indicating that a blockade of TIM3 could not only increase antitumor CD8 T cells activation and IFN-γ secretion, but would also favour cDC1 cells and CD8 T cells to colocalize within the tumour, amplifying the chance to mount an appropriate antitumor response both in time and space (Gardner et al., 2022). Moreover, it has been very recently described that BAT3 is a specific and endogenous regulator of tolerogenic DCs phenotype and function able to favour a pro-tumour T cell compartment (reduction of TH1 cells, and increase in Treg cells) (Tang et al., 2022). Finally, in term of metabolic activity, it is now clear that TIM3 and Gal9 act as enhancer of immunoregulatory function through a shift in glycolysis at the expense of OXPHOS. This has been well described for Treg cells (Banerjee et al., 2021) and due to well established knowledge about tolerogenic DCs, one can speculate the same feature for them (Dáňová et al., 2015). Altogether, these studies indicate clearly the major role displayed by the Gal9/TIM3 axis, both in CIS and TRANS, in the emergence of regulatory DCs, notably within the tumour microenvironment. Indeed, one can resume that Gal9 binding to TIM3 on DC reinforce its membrane stability, allows TIM3 to oligomerize in order to deliver its immunoregulatory signal. This signal relies both on the degradation of STING and on the release of BAT3 protective adapter from the cytoplasmic tail of TIM3. These two complementary signals lead to the activation of STA3 and NFκB transcription factors resulting in blocking IL-12 secretion in favour of IL-10 and IL-6 release (Barton, 2006), such as described for tumour cells (Zhang et al., 2018). Because of this dysregulation, Dendritic Cells lose their ability to activate antitumor T cells response and rather facilitate tolerance in T cells via multiple mechanisms already described elsewhere such as T cell anergy, T cell depletion or conversion into FOXP3 regulatory T cells (Iberg et al., 2017).

In the present study, we described the emergence of tolerogenic mregDCs after exposure to Gal9+ NPC derived SEVs. Indeed, all the event described above are representative and perfectly in line with what could happen to DCs during their development in the presence of NPC SEVs in the tumour context, and the consequence in term of regulatory gene overexpression (Supplementary data 6 B and D), metabolism shift, and both cytokine release and T cell anergy is strongly in favour of the role of Gal9/TIM3 axis so. Nevertheless, to prove indisputably the role of Gal9 on this process, we decided to move further and use a specific mAb able to block its capacities as we described with our collaborators (Lhuillier et al., 2018; Sarahjibrin, 2021). Our results indicate that this antibody is able to block T cell death or anergy triggered by recombinant Galectine-9. In the present work, we used this original anti-Gal9 blocking antibody (1g3) that allows us to prove that the immunosuppressive Gal9 could partly be responsible for the change in the fate of DC in its secreted form but overall associated to extracellular vesicles. This indication reinforces our understanding of the immunosuppressive role played by Gal9 itself in NPC and the ability of EVs to convey this property on target cells.

Finally, in parallel to previous work from the team demonstrating that NPC-Exo could recruit Tregs at the tumour site both In vitro and in vivo (Mrizak et al., 2015), we intend to demonstrate that NPCSEVs preferentially attract NPCSEV-DC which could also enhance the suppressive microenvironment present in NPC. Interestingly we observed that tDC do not migrate under any chemotactic gradient as shown in the migration movie of tDC under a C15SEV gradient (Supplementary data 1). This could be explained by the use of vitamin D3 and dexamethasone to generate them. Indeed, another study reports similar results with tDC generated with vitamin D3 or dexamethasone that do not migrate toward a CCL19 gradient and have an under-expression of CCR7, the CCL19 receptor (Adnan et al., 2016; Barragan et al., 2015). It is important to note that glucocorticoids as dexamethasone are able to cause microtubule and F-actin rearrangement in T lymphocytes leading to depolarization of the cells preventing their migration (Müller et al., 2013). Same action was described in mesenchymal stem cell that loss migration behaviour after dexamethasone treatment (Schneider et al., 2015) and could have led to the paralytic state of tDC that we observed.

Given that the presence of conventional DCs can be considered very early in tumour development and given the role of mregDCs in resistance to anti-cancer treatments, it can be assumed that SEVs are one of the main players in this feature. Above immunotherapies, this cellular mechanism could probably explain, at least in part, the fatal resistance observed in too many NPC patients after recurrence of the classical radio-chemo therapeutic approach.

Altogether our data, offers a new point of view about the role of NPC derived SEV on DCs. This study presents for the first time that tumour derived small extracellular vesicles contribute to NPC immune evasion via the emergence of mregDCs through Gal9. This indirect mechanism enhances the lack of proinflammatory cytokines production and decrease capacity to activate effector
T cells probably via IL-4 production, and thus contribute to the immunosuppressive tumour microenvironment. These results coincide with a single cell study in NPC that described the presence of mregDCs into NPC microenvironment (Chen et al., 2020) and with TCGA dataset in H&N cancer. Tumour derived small extracellular vesicles are major players in tumorigenesis and of great interest as therapeutic targets then. We are now one-step closer to decipher the immune evasion mechanisms put in place by the tumour although there are many others still to discover. At this time of great promise for anticancer immunotherapies, better understanding tumour immune evasion will help us to improved immunotherapy and develop new immunotherapeutic drugs.

## AUTHOR CONTRIBUTIONS

**Anthony Lefebvre:** Formal analysis; investigation; methodology; software; writing—original draft; writing—review and editing. **Camille Trioën:** Formal analysis; investigation; methodology; writing—original draft; writing—review and editing. **Sarah Renaud:** Investigation; methodology; writing—original draft. **William Laine:** Investigation. **Benjamin Hennart:** Formal analysis; methodology; resources; software. **Clément Bouchez:** Investigation. **Bertrand Leroux:** Investigation; methodology. **Delphine Allorge:** Methodology; resources. **Jérôme Kluza:** Methodology; resources; validation. **Guillaume Paul Grolez:** Formal analysis; investigation. **Nadira Delhem:** Conceptualization; funding acquisition; project administration; validation; writing—review and editing

## ACKNOWLEDGEMENTS

The authors would like to thank both the BioImaging Center Lille campus Pasteur for access to flow cytometry equipment. The authors would also like to thanks the Animal Experimentation and High Technology Platform (PLEHTA) at Lille campus Pasteur for access to equipment. This research program was supported by grant from Canceropôle Nord OUEST Project CNO2019. N. Delhem and O. Moralès have received a Grant from the Canceropôle Nord-Ouest. Emerging Project CNO2014.

## CONFLICT OF INTEREST STATEMENT

The authors declare no conflict of interest.

## ORCID

Olivier Moralès https://orcid.org/0000-0002-2135-3265

## REFERENCES

Adnan, E., Matsumoto, T., Ishizaki, J., Onishi, S., Suemori, K., Yasukawa, M., & Hasegawa, H. (2016). Human tolerogenic dendritic cells generated with protein kinase c inhibitor are optimal for functional regulatory T cell induction—A comparative study. *Clinical Immunology* (Orlando, Fla.), **173**, 96–108. https://doi.org/10.1016/j.clim.2016.09.007

Anderson, A. C., Anderson, D. E., Bregoli, L., Hastings, W. D., Kassam, N., Lei, C., Chandwaskar, R., Karman, J., Su, E. W., Hirashima, M., Bruce, J. N., Kane, L. P., Kuchroo, V. K., & Hafler, D. A. (2007). Promotion of tissue inflammation by the immune receptor Tim-3 expressed on innate immune cells. *Science* (New York, N.Y.), **318**(5853), 1141–1143. https://doi.org/10.1126/science.1148536

Banerjee, H., Nieves-Rosado, H., Kulkarni, A., Murter, B., McGrath, K. V., Chandran, U. R., Chang, A., Szymczak-Workman, A. L., Vujanovic, L., Delgoffe, G. M., Ferris, R. L., & Kane, L. P. (2021). Expression of Tim-3 drives phenotypic and functional changes in Treg cells in secondary lymphoid organs and the tumor microenvironment. *Cell Reports*, **36**(11), 10969. https://doi.org/10.1016/j.celrep.2021.109699

Barragan, M., Good, M., & Kolls, J. K. (2015). Regulation of dendritic cell function by vitamin D. *Nutrients*, **7**(9), 8127–8151. https://doi.org/10.3390/nu7095383

Barton, B. E. (2006). STAT3: A potential therapeutic target in dendritic cells for the induction of transplant tolerance. *Expert Opinion on Therapeutic Targets*, **10**(3), 459–470. https://doi.org/10.1517/14728222.10.3.459

Bei, J.-X., Jia, W.-H., & Zeng, Y.-X. (2012). Familial and large-scale case-control studies identify genes associated with nasopharyngeal carcinoma. *Seminars in Cancer Biology*, **22**(2), 96–106. https://doi.org/10.1016/j.semcancer.2012.01.012

Belladonna, M. L., Grohmann, U., Guidetti, P., Volpi, C., Bianchi, R., Fioretti, M. C., Schwarcz, R., Fallarino, F., & Puccetti, P. (2006). Kynurenine pathway enzymes in dendritic cells initiate tolerogenesis in the absence of functional IDO. *Journal of Immunology* (Baltimore, Md.: 1950), **177**(1), 130–137. https://doi.org/10.4049/jimmunol.177.1.130

Boulland, M.-L., Marquet, J., Molinier-Frenkel, V., Möller, P., Guiter, C., Lasoudris, F., Copie-Bergman, C., Baia, M., Gaulard, P., Leroy, K., & Castellano, F. (2007). Human IL4I1 is a secreted L-phenylalanine oxidase expressed by mature dendritic cells that inhibits T-lymphocyte proliferation. *Blood*, **110**(1), 220–227. https://doi.org/10.1182/blood-2006-07-036210

Budiani, D. R., Haryana, S. M., & Sosroseno, W. (2011). Interleukin-4 and interferon-γ levels in Epstein-Barr virus-associated infectious mononucleosis and nasopharyngeal carcinoma. *Journal of Research in Medical Sciences: The Official Journal of Isfahan University of Medical Sciences*, **16**(1), 94–97.

Chang, E. T., & Adami, H.-O. (2006). The enigmatic epidemiology of nasopharyngeal carcinoma. *Cancer Epidemiology, Biomarkers & Prevention: A Publication of the American Association for Cancer Research, Cosponsored by the American Society of Preventive Oncology*, 15(10), 1765–1777. https://doi.org/10.1158/1055-9965.EPI-06-0353

Chaves, A. C., Cerávolo, I. P., Gomes, J. A., Zani, C. L., Romanha, A. J., & Gazzinelli, R. T. (2001). IL-4 and IL-13 regulate the induction of indoleamine 2,3-dioxygenase activity and the control of Toxoplasma gondii replication in human fibroblasts activated with IFN-gamma. *European Journal of Immunology*, **31**(2), 333–344. https://doi.org/10.1002/1521-4141(200102)31:2<333::aid-immu333>3.0.co;2-x

Chen, T.-C., Chen, C.-H., Wang, C.-P., Lin, P.-H., Yang, T.-L., Lou, P.-J., Ko, J.-Y., Wu, C.-T., & Chang, Y.-L. (2017). The immunologic advantage of recurrent nasopharyngeal carcinoma from the viewpoint of Galectin-9/Tim-3-related changes in the tumour microenvironment. *Scientific Reports*, **7**(1), 10349. https://doi.org/10.1038/s41598-017-10386-y
T cells probably via IL-4 production, and thus contribute to the immunosuppressive tumour microenvironment. These results coincide with a single cell study in NPC that described the presence of mregDCs into NPC microenvironment (Chen et al., 2020) and with TCGA dataset in H&N cancer. Tumour derived small extracellular vesicles are major players in tumorigenesis and of great interest as therapeutic targets then. We are now one-step closer to decipher the immune evasion mechanisms put in place by the tumour although there are many others still to discover. At this time of great promise for anticancer immunotherapies, better understanding tumour immune evasion will help us to improved immunotherapy and develop new immunotherapeutic drugs.

## AUTHOR CONTRIBUTIONS

**Anthony Lefebvre:** Formal analysis; investigation; methodology; software; writing—original draft; writing—review and editing. **Camille Trioën:** Formal analysis; investigation; methodology; writing—original draft; writing—review and editing. **Sarah Renaud:** Investigation; methodology; writing—original draft. **William Laine:** Investigation. **Benjamin Hennart:** Formal analysis; methodology; resources; software. **Clément Bouchez:** Investigation. **Bertrand Leroux:** Investigation; methodology. **Delphine Allorge:** Methodology; resources. **Jérôme Kluza:** Methodology; resources; validation. **Guillaume Paul Grolez:** Formal analysis; investigation. **Nadira Delhem:** Conceptualization; funding acquisition; project administration; validation; writing—review and editing

## ACKNOWLEDGEMENTS

The authors would like to thank both the BioImaging Center Lille campus Pasteur for access to flow cytometry equipment. The authors would also like to thanks the Animal Experimentation and High Technology Platform (PLEHTA) at Lille campus Pasteur for access to equipment. This research program was supported by grant from Canceropôle Nord OUEST Project CNO2019. N. Delhem and O. Moralès have received a Grant from the Canceropôle Nord-Ouest. Emerging Project CNO2014.

## CONFLICT OF INTEREST STATEMENT

The authors declare no conflict of interest.

## ORCID

Olivier Moralès https://orcid.org/0000-0002-2135-3265

## REFERENCES

Adnan, E., Matsumoto, T., Ishizaki, J., Onishi, S., Suemori, K., Yasukawa, M., & Hasegawa, H. (2016). Human tolerogenic dendritic cells generated with protein kinase c inhibitor are optimal for functional regulatory T cell induction—A comparative study. *Clinical Immunology* (Orlando, Fla.), **173**, 96–108. https://doi.org/10.1016/j.clim.2016.09.007

Anderson, A. C., Anderson, D. E., Bregoli, L., Hastings, W. D., Kassam, N., Lei, C., Chandwaskar, R., Karman, J., Su, E. W., Hirashima, M., Bruce, J. N., Kane, L. P., Kuchroo, V. K., & Hafler, D. A. (2007). Promotion of tissue inflammation by the immune receptor Tim-3 expressed on innate immune cells. *Science* (New York, N.Y.), **318**(5853), 1141–1143. https://doi.org/10.1126/science.1148536

Banerjee, H., Nieves-Rosado, H., Kulkarni, A., Murter, B., McGrath, K. V., Chandran, U. R., Chang, A., Szymczak-Workman, A. L., Vujanovic, L., Delgoffe, G. M., Ferris, R. L., & Kane, L. P. (2021). Expression of Tim-3 drives phenotypic and functional changes in Treg cells in secondary lymphoid organs and the tumor microenvironment. *Cell Reports*, **36**(11), 10969. https://doi.org/10.1016/j.celrep.2021.109699

Barragan, M., Good, M., & Kolls, J. K. (2015). Regulation of dendritic cell function by vitamin D. *Nutrients*, **7**(9), 8127–8151. https://doi.org/10.3390/nu7095383

Barton, B. E. (2006). STAT3: A potential therapeutic target in dendritic cells for the induction of transplant tolerance. *Expert Opinion on Therapeutic Targets*, **10**(3), 459–470. https://doi.org/10.1517/14728222.10.3.459

Bei, J.-X., Jia, W.-H., & Zeng, Y.-X. (2012). Familial and large-scale case-control studies identify genes associated with nasopharyngeal carcinoma. *Seminars in Cancer Biology*, **22**(2), 96–106. https://doi.org/10.1016/j.semcancer.2012.01.012

Belladonna, M. L., Grohmann, U., Guidetti, P., Volpi, C., Bianchi, R., Fioretti, M. C., Schwarcz, R., Fallarino, F., & Puccetti, P. (2006). Kynurenine pathway enzymes in dendritic cells initiate tolerogenesis in the absence of functional IDO. *Journal of Immunology* (Baltimore, Md.: 1950), **177**(1), 130–137. https://doi.org/10.4049/jimmunol.177.1.130

Boulland, M.-L., Marquet, J., Molinier-Frenkel, V., Möller, P., Guiter, C., Lasoudris, F., Copie-Bergman, C., Baia, M., Gaulard, P., Leroy, K., & Castellano, F. (2007). Human IL4I1 is a secreted L-phenylalanine oxidase expressed by mature dendritic cells that inhibits T-lymphocyte proliferation. *Blood*, **110**(1), 220–227. https://doi.org/10.1182/blood-2006-07-036210

Budiani, D. R., Haryana, S. M., & Sosroseno, W. (2011). Interleukin-4 and interferon-γ levels in Epstein-Barr virus-associated infectious mononucleosis and nasopharyngeal carcinoma. *Journal of Research in Medical Sciences: The Official Journal of Isfahan University of Medical Sciences*, **16**(1), 94–97.

Chang, E. T., & Adami, H.-O. (2006). The enigmatic epidemiology of nasopharyngeal carcinoma. *Cancer Epidemiology, Biomarkers & Prevention: A Publication of the American Association for Cancer Research, Cosponsored by the American Society of Preventive Oncology*, 15(10), 1765–1777. https://doi.org/10.1158/1055-9965.EPI-06-0353

Chaves, A. C., Cerávolo, I. P., Gomes, J. A., Zani, C. L., Romanha, A. J., & Gazzinelli, R. T. (2001). IL-4 and IL-13 regulate the induction of indoleamine 2,3-dioxygenase activity and the control of Toxoplasma gondii replication in human fibroblasts activated with IFN-gamma. *European Journal of Immunology*, **31**(2), 333–344. https://doi.org/10.1002/1521-4141(200102)31:2<333::aid-immu333>3.0.co;2-x

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Clayton, K. L., Haaland, M. S., Douglas-Vail, M. B., Mujib, S., Chew, G. M., Ndhlovu, L. C., & Ostrowski, M. A. (2014). T cell Ig and mucin domain-containing protein 3 is recruited to the immune synapse, disrupts stable synapse formation, and associates with receptor phosphatases. *Journal of Immunology (Baltimore, Md.: 1950)*, **192**(2), 782–791. https://doi.org/10.4049/jimmunol.1302663

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de Mingo Pulido, Á., Hänggi, K., Celias, D. P., Gardner, A., Li, J., Batista-Bittencourt, B., Mohamed, E., Trillo-Tinoco, J., Osunmakinde, O., Peña, R., Onimus, A., Kaisho, T., Kaufmann, J., McEachern, K., Soliman, H., Luca, V. C., Rodriguez, P. C., Yu, X., & Ruffell, B. (2021). The inhibitory receptor TIM-3 limits activation of the cGAS-STING pathway in intra-tumoral dendritic cells by suppressing extracellular DNA uptake. *Immunity*, **54**(6), 1154–1167.e7. https://doi.org/10.1016/j.immuni.2021.04.019

DeVito, N. C., Plebanek, M. P., Theivanthiran, B., & Hanks, B. A. (2019). Role of tumor-mediated dendritic cell tolerization in immune evasion. *Frontiers in Immunology*, **10**, 2876. https://doi.org/10.3389/fimmu.2019.02876

Dons, E. M., Raimondi, G., Cooper, D. K. C., & Thomson, A. W. (2012). Induced regulatory T cells: Mechanisms of conversion and suppressive potential. *Human Immunology*, **73**(4), 328–334. https://doi.org/10.1016/j.humimm.2011.12.011

Dukers, D. F., Meij, P., Vervoort, M. B., Vos, W., Scheper, R. J., Meijer, C. J., Bloemena, E., & Middeldorp, J. M. (2000). Direct immunosuppressive effects of EBV-encoded latent membrane protein 1. *Journal of Immunology (Baltimore, Md.: 1950)*, **165**(2), 663–670. https://doi.org/10.4049/jimmunol.165.2.663

Duvallet, E., Boulpicante, M., Yamazaki, T., Daskalogianni, C., Martins, R. P., Baconnais, S., Manoury, B., Fahraeus, R., & Apcher, S. (2016). Exosome-driven transfer of tumor-associated pioneer translation products (TA-PTPs) for the MHC class I cross-presentation pathway. *Oncoimmunology*, **5**(9), e1198865. https://doi.org/10.1080/2162402X.2016.1198865

Enrica, F., Andrea, C., Cristiana, C., Mirella, G., Costanza, A., Paolo, C.-P., Ciro, T., Giovani, C., & Maria, M. Z. (2016). Human mesenchymal stem cells and derived extracellular vesicles induce regulatory dendritic cells in type 1 diabetic patients. *Diabetologia*, **59**(2), 325–333. https://doi.org/10.1007/s00125-015-3808-0

Fujita, K., Iwama, H., Sakamoto, T., Okura, R., Kobayashi, K., Takano, J., Katsura, A., Tatsuta, M., Maeda, E., Mimura, S., Nomura, T., Tani, J., Miyoshi, H., Morishita, A., Yoneyama, H., Yamana, Y., Himoto, T., Okano, K., Suzuki, Y., ... Masaki, T. (2015). Galectin-9 suppresses the growth of hepatocellular carcinoma via apoptosis in vitro and in vivo. *International Journal of Oncology*, **46**(6), 2419–2430. https://doi.org/10.3892/ijo.2015.2941

Gardner, A., Pulido, Á. M., Hänggi, K., Bazargan, S., Onimus, A., Kasprzak, A., Conejo-Garcia, J. R., Rejniski, K. A., & Ruffell, B. (2022). TIM-3 blockade enhances IL-12-dependent antitumor immunity by promoting CD8+ T cell and XCR1+ dendritic cell spatial co-localization. *Journal for Immunotherapy of Cancer*, **10**(1), e003571. https://doi.org/10.1136/jitc-2021-003571

Gargaro, M., Scalisi, G., Manni, G., Briseño, C. G., Bagadia, P., Durai, V., Theisen, D. J., Kim, S., Castelli, M., Xu, C. A., Meyer Zu Hörste, G., Servillo, G., Della Fazia, M. A., Mencarelli, G., Ricciuti, D., Padiglioni, E., Giacchè, N., Colliva, C., Pellicciari, R., ... Fallarino, F. (2022). Indoleamine 2,3-dioxygenase 1 activation in mature cDC1 promotes tolerogenic education of inflammatory cDC2 via metabolic communication. *Immunity*, **55**(6), 1032–1050.e14. https://doi.org/10.1016/j.immuni.2022.05.013

Gourzones, C., Barjon, C., & Busson, P. (2012). Host-tumor interactions in nasopharyngeal carcinomas. *Seminars in Cancer Biology*, **22**(2), 127–136. https://doi.org/10.1016/j.semcancer.2012.01.002

Harden, J. L., & Egilmez, N. K. (2012). Indoleamine 2,3-dioxygenase and dendritic cell tolerogenicity. *Immunological Investigations*, **41**(6–7), 738–764. https://doi.org/10.3109/08820139.2012.676122

Huang, X., Li, T., Chen, J., Wang, Y., Chen, S., Deng, W., & Huang, Q. (2020). AB0693 Tim-3-expressing neutrophils as a novel indicator to assess disease activity and severity in ankylosing spondylitis. *Annals of the Rheumatic Diseases*, **79**(Suppl 1), 1641–1642. https://doi.org/10.1136/annrheumdis-2020-ular.5466

Iberg, C. A., Jones, A., & Hawiger, D. (2017). Dendritic cells as inducers of peripheral tolerance. *Trends in Immunology*, **38**(11), 793–804. https://doi.org/10.1016/j.it.2017.07.007

Keryer-Bibens, C., Pioche-Durieu, C., Villemant, C., Souquère, S., Nishi, N., Hirashima, M., Middeldorp, J., & Busson, P. (2006). Exosomes released by EBV-infected nasopharyngeal carcinoma cells convey the viral latent membrane protein 1 and the immunomodulatory protein galectin 9. *BMC Cancer*, **6**, 283. https://doi.org/10.1186/1471-2407-6-283

Kharaziha, P., Ceder, S., Li, Q., & Panaretakis, T. (2012). Tumor cell-derived exosomes: A message in a bottle. *Biochimica et Biophysica Acta (BBA)—Reviews on Cancer*, **1826**(1), 103–111. https://doi.org/10.1016/j.bbcan.2012.03.006

Kim, S. H., Jung, H. H., & Lee, C. K. (2018). Generation, characteristics and clinical trials of ex vivo generated tolerogenic dendritic cells. *Yonsei Medical Journal*, **59**(7), 807–815. https://doi.org/10.3349/ymj.2018.59.7.807

Klibi, J., Niki, T., Riedel, A., Pioche-Durieu, C., Souquere, S., Rubinstein, E., Le Moulec, S., Guigay, J., Hirashima, M., Guemira, F., Adhikary, D., Mautner, J., & Busson, P. (2009). Blood diffusion and Th1-suppressive effects of Galectin-9-containing exosomes released by Epstein-Barr virus-infected nasopharyngeal carcinoma cells. *Blood*, **113**(9), 1957–1966. https://doi.org/10.1182/blood-2008-02-142596

Kobayashi, M., Kobayashi, H., Pollard, R. B., & Suzuki, F. (1998). A pathogenic role of Th2 cells and their cytokine products on the pulmonary metastasis of murine B16 melanoma. *Journal of Immunology (Baltimore, Md.: 1950)*, **160**(12), 5869–5873.

Krause, P., Singer, E., Darley, P. I., Klebensberger, J., Groettrup, M., & Legler, D. F. (2007). Prostaglandin E2 is a key factor for monocyte-derived dendritic cell maturation: Enhanced T cell stimulatory capacity despite IDO. *Journal of Leukocyte Biology*, **82**(5), 1106–1114. https://doi.org/10.1189/jlb.0905519

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## SUPPORTING INFORMATION

Additional supporting information can be found online in the Supporting Information section at the end of this article.

**How to cite this article:** Lefebvre, A., Trioën, C., Renaud, S., Laine, W., Hennart, B., Bouchez, C., Leroux, B., Allorge, D., Kluza, J., Werkmeister, E., Grolez, G. P., Delhem, N., & Moralès, O. (2023). Extracellular Vesicles derived from Nasopharyngeal Carcinoma induce the Emergence of Mature Regulatory Dendritic Cells using a Galectin-9 dependent mechanism. *Journal of Extracellular Vesicles*, 12, e12390. https://doi.org/10.1002/jev2.12390
Wang, J., De Veirman, K., De Beule, N., Maes, K., De Bruyne, E., Van Valckenborgh, E., Vanderkerken, K., & Menu, E. (2015). The bone marrow microenvironment enhances multiple myeloma progression by exosome-mediated activation of myeloid-derived suppressor cells. *Oncotarget*, 6(41), 43992-44004. https://doi.org/10.18632/oncotarget.6083

Weyer, P. S., Muehlfeit, M., Klose, C., Bonventre, J. V., Walz, G., & Wolfgang Kuehn, E. (2006). A highly conserved tyrosine of Tim-3 is phosphorylated upon stimulation by its ligand galectin-9. *Biochemical and Biophysical Research Communications*, 351(2), 571-576. https://doi.org/10.1016/j.bbrc.2006.10.079

Wolf, Y., Anderson, A. C., & Kuchroo, V. K. (2020). TIM3 comes of age as an inhibitory receptor. *Nature Reviews. Immunology*, 20(3), 173-185. https://doi.org/10.1038/s41577-019-0224-6

Wu, C., Thalhamer, T., Franca, R. F., Xiao, S., Wang, C., Hotta, C., Zhu, C., Hirashima, M., Anderson, A. C., & Kuchroo, V. K. (2014). Galectin-9-CD44 interaction enhances stability and function of adaptive regulatory T cells. *Immunity*, 41(2), 270-282. https://doi.org/10.1016/j.immuni.2014.06.011

Wu, L., Li, C., & Pan, L. (2018). Nasopharyngeal carcinoma: A review of current updates. *Experimental and Therapeutic Medicine*, 15(4), 3687-3692. https://doi.org/10.3892/etm.2018.5878

Yamauchi, A., Dai, S. Y., Nakagawa, R., Kashio, Y., Abe, H., Katoh, S., Kontani, K., & Hirashima, M. (2005). Galectin-9 induces maturation of human monocyte-derived dendritic cells. *Journal of Immunology (Baltimore, Md.: 1950)*, 175(5), 2974-2981. https://doi.org/10.4049/jimmunol.175.5.2974

Ye, S.-B., Li, Z.-L., Luo, D.-H., Huang, B.-J., Chen, Y.-S., Zhang, X.-S., Cui, J., Zeng, Y.-X., & Li, J. (2014). Tumor-derived exosomes promote tumor progression and T-cell dysfunction through the regulation of enriched exosomal microRNAs in human nasopharyngeal carcinoma. *Oncotarget*, 5(14), 5439-5452. https://doi.org/10.18632/oncotarget.2118

Yip, W. K., Abdullah, M. A., Yusoff, S. M., & Seow, H. F. (2009). Increase in tumour-infiltrating lymphocytes with regulatory T cell immunophenotypes and reduced zeta-chain expression in nasopharyngeal carcinoma patients. *Clinical and Experimental Immunology*, 155(3), 412-422. https://doi.org/10.1111/j.1365-2249.2008.03793.x

Young, L. S., & Rickinson, A. B. (2004). Epstein-Barr virus: 40 years on. *Nature Reviews. Cancer*, 4(10), 757-768. https://doi.org/10.1038/nrc1452

Zeyda, M., Säemann, M. D., Stuhlmeier, K. M., Mascher, D. G., Nowotny, P. N., Zlabinger, G. J., Waldhäusl, W., & Stulnig, T. M. (2005). Polyunsaturated Fatty acids block dendritic cell activation and function independently of NF-kappaB activation. *The Journal of Biological Chemistry*, 280(14), 14293-14301. https://doi.org/10.1074/jbc.M410000200

Zhang, C.-X., Huang, D.-J., Baloche, V., Zhang, L., Xu, J.-X., Li, B.-W., Zhao, X.-R., He, J., Mai, H. Q., Chen, Q. Y., Zhang, X. S., Busson, P., Cui, J., & Li, J. (2020). Galectin-9 promotes a suppressive microenvironment in human cancer by enhancing STING degradation. *Oncogenesis*, 9(7), 65. https://doi.org/10.1038/s41389-020-00248-0

Zhang, H., Song, Y., Yang, H., Liu, Z., Gao, L., Liang, X., & Ma, C. (2018). Tumor cell-intrinsic Tim-3 promotes liver cancer via NF-κB/IL-6/STAT3 Axis. *Oncogene*, 37(18), 2456-2468. https://doi.org/10.1038/s41388-018-0140-4

Zhang, W., Zhang, Y., He, Y., Wang, X., & Fang, Q. (2019). Lipopolysaccharide mediates time-dependent macrophage M1/M2 polarization through the Tim-3/Galectin-9 signalling pathway. *Experimental Cell Research*, 376(2), 124-132. https://doi.org/10.1016/j.yexcr.2019.02.007

Zhou, X., Sun, L., Jing, D., Xu, G., Zhang, J., Lin, L., Zhao, J., Yao, Z., & Lin, H. (2018). Galectin-9 expression predicts favorable clinical outcome in solid tumors: A systematic review and meta-analysis. *Frontiers in Physiology*, 9, 452. https://doi.org/10.3389/fphys.2018.00452

Zhu, C., Anderson, A. C., Schubart, A., Xiong, H., Imitola, J., Khoury, S. J., Zheng, X. X., Strom, T. B., & Kuchroo, V. K. (2005). The Tim-3 ligand Galectin-9 negatively regulates T Helper type 1 immunity. *Nature Immunology*, 6(12), 1245-1252. https://doi.org/10.1038/ni1271

Zhu, W., Stevens, A. P., Dettmer, K., Gottfried, E., Hoves, S., Kreutz, M., Holler, E., Canelas, A. B., Kema, I., & Oefner, P. J. (2011). Quantitative profiling of tryptophan metabolites in serum, urine, and cell culture supernatants by liquid chromatography-tandem mass spectrometry. *Analytical and Bioanalytical Chemistry*, 401(10), 3249-3261. https://doi.org/10.1007/s00216-011-5436-y

Zilionis, R., Engblom, C., Pfirschke, C., Savova, V., Zemmour, D., Saatcioglu, H. D., Krishnan, I., Maroni, G., Meyerovitz, C. V., Kerwin, C. M., Choi, S., Richards, W. G., De Rienzo, A., Tenen, D. G., Bueno, R., Levantini, E., Pittet, M. J., & Klein, A. M. (2019). Single-cell transcriptomics of human and mouse lung cancers reveals conserved myeloid populations across individuals and species. *Immunity*, 50(5), 1317-1334.e10. https://doi.org/10.1016/j.immuni.2019.03.009

## SUPPORTING INFORMATION

Additional supporting information can be found online in the Supporting Information section at the end of this article.

**How to cite this article:** Lefebvre, A., Trioën, C., Renaud, S., Laine, W., Hennart, B., Bouchez, C., Leroux, B., Allorge, D., Kluza, J., Werkmeister, E., Grolez, G. P., Delhem, N., & Moralès, O. (2023). Extracellular Vesicles derived from Nasopharyngeal Carcinoma induce the Emergence of Mature Regulatory Dendritic Cells using a Galectin-9 dependent mechanism. *Journal of Extracellular Vesicles*, 12, e12390. https://doi.org/10.1002/jev2.12390