Immuno-subversion of BDCA1+ cDC2s, BDCA2+ pDCs and BDCA3+ cDC1s dendritic cell subsets in the context of melanoma : implication of the glycan / C-type lectin receptor axis

La subversion de la réponse immunitaire par les tumeurs est une étape cruciale pour leur développement. Les cellules dendritiques (DCs), composées des sous-types BDCA1+ cDC2s, BDCA2+ pDCs et BDCA3+ cDC1s, sont essentielles pour l'initiation et l'orientation des réponses immunes anti-tumorales. Cependant, les cellules tumorales subvertissent leurs fonctions, en exploitant leur plasticité, afin d'échapper au contrôle du système immunitaire. L'origine de cette subversion n'a pas encore été totalement élucidée. Les cellules tumorales possèdent à leur surface une glycosylation aberrante des glycoprotéines et glycolipides qui peut être reconnue par les récepteurs lectine de type C (CLR), exprimés par les DCs et cruciaux pour déclencher et orienter les réponses subséquentes. Néanmoins, le rôle pathophysiologique des sous-types de DCs et leurs profils d'expression des CLRs reste largement inconnu en contexte du mélanome. De plus, le glyco-code tumoral et son impact sur l'immunité est encore à élucider.Nous avons examiné, chez des patients atteints d'un mélanome, les caractéristiques phénotypiques (molécules de co-stimulation, CLRs) et fonctionnelles des sous-types de DCs circulantes ou infiltrant la tumeur. Nous avons évalué l'impact clinique de ces observations et les corrélations entre les profils d'expression des CLRs et les caractéristiques des DCs. Afin de décrypter le lien potentiel entre les profils aberrants de glycosylation et l'évasion immunitaire en contexte du mélanome, nous avons étudié le glyco-code des cellules tumorales dérivées de patients grâce à la technologie GLYcoPROFILE™ et étudié sa relevance clinique et son impact sur la fonctionnalité des DCs.Les sous-types de DCs présentent des fréquences perturbées dans le sang et infiltrent la tumeur. Les cDC2s et pDCs exhibent une fonctionnalité altérée en réponse à la stimulation TLR, tandis que les cDC1s (circulantes ou infiltrant la tumeur) préservent leur compétences anti-tumorales et sont associées avec un meilleur pronostic des patients. De plus, nous avons pu mettre en évidence une forte modulation du répertoire des CLRs des sous-types de DCs circulantes ou infiltrant la tumeur dans les patients atteint d'un mélanome. En effet, les expressions de DCIR, Clec12α et NKp44 sont modulées sur les DCs circulantes, tandis que les niveaux de Dectin-1, CD206, DEC205, DC-SIGN et Clec9α sont altérés dans les DCs infiltrant la tumeur. De façon intéressante, des fortes proportions de cDC1s circulantes et de cDCs exprimant DCIR, DEC205 et Clec12α sont corrélées avec une meilleure survie des patients. D'autre part, de fortes proportions de cDCs CD206+ ou Dectin1+ et pDCs NKp44+ étaient associées avec un mauvais impact clinique. De plus, l'étude du glyco-code des cellules tumorales a révélé que les motifs GlcNAc, NeuAc, TF-Ag et Fuc sont associés avec un mauvais pronostic clinique, tandis que Man et Glc ont entraîné une meilleure survie. Nous avons aussi pu observer que les cellules tumorales ayant un glyco-code distinct impactaient différemment la production de cytokines par les sous-types de DCs. Une forte expression de GlcNAc avait une influence négative sur les cDC2s, tandis que de forts niveaux des motifs Fuc et Gal impactaient négativement les cDC1s et pDCs. Le blocage spécifique de certains glycans a permis de restaurer la fonctionnalité des DCs. Le glyco-code tumoral était aussi lié avec la nature et densité de l'infiltrat.Cette étude révèle l'impact critique des sous-types de DCs sur le pronostic clinique et l'effet des glycans sur l'immunité, et suggère que le mélanome peut manipuler les CLRs pour détourner les DCs et échapper au contrôle immunitaire. Les interactions glycans-CLR émergent comme de nouveaux points de contrôle immunitaires, en contexte de mélanome, permettant de restaurer la subversion des DCs afin de rétablir une réponse anti-tumorale efficace et d'inhiber les voies immunosuppressives déclenchées par la glycosylation aberrante des tumeurs.Subversion of immunity is a hallmark of cancer development. Dendritic cells (DCs) are strategic immune cells, composed of specialized subsets (cDC1s, cDC2s, pDCs), that initiate and shape anti-tumor immune responses, but tumor cells exploit their plasticity to subvert their functions and escape immune control. The bases for such DCs' hijacking are yet to be fully elucidated. Tumor cells harbor unusual glycosylation patterns of surface glycoproteins and glycolipids that can be sensed through C-type lectin receptors (CLR), expressed on DCs, and subsequently trigger and orientate antitumor immunity. Yet, the pathophysiological role of DCs subsets and the status of their CLR machinery remained largely unknown in the context of melanoma. Furthermore, the global tumor glyco-code and its impact on immunity remains to be elucidated.We investigated the phenotypic (co-stimulatory molecules, CLR patterns) and functional features of circulating and tumor-infiltrating BDCA1+ cDC2s, BDCA2+ pDCs, BDCA3+ cDC1s of melanoma patients, assessed their clinical impact, and further depicted the correlations between CLR expression profiles and other DCs' features. Moreover, to decrypt the potential link between aberrant glycosylation patterns and immune evasion in melanoma, we investigated the melanoma tumor glyco-code through the GLYcoPROFILE™ methodology (lectin arrays), and depicted its impact on patients' clinical outcome and DC subsets' functionality.DC subsets exhibited perturbed frequencies in the circulation and actively infiltrated the tumor site, while harboring a higher activation status. Whereas cDC2s and pDCs displayed an altered functionality in response to TLR-triggering, circulating and tumor-infiltrating cDC1s preserved potent competences associated with improved prognosis. We further highlighted that the CLR repertoire of circulating and tumor-infiltrating cDC1s, cDC2s, and pDCs was strongly perturbed in melanoma patients, with modulation of DCIR, CLEC-12α and NKp44 on circulating DCs, and perturbation of Dectin-1, CD206, DEC205, DC-SIGN and CLEC-9α on tumor-infiltrating DCs. Notably, higher frequencies of circulating cDC1s and higher proportions of DCIR-, DEC205-, CLEC-12α-expressing cDCs were linked with a better clinical outcome of melanoma patients. On the other hand, elevated proportions of CD206-, Dectin1-expressing cDCs and NKp44-expressing pDCs were associated with a poor outcome. Furthermore, when studying the melanoma glyco-code, specific glycan patterns correlated with clinical outcome of melanoma patients, GlcNAc, NeuAc, TF-Ag and Fuc motifs being associated with poor outcome, whereas Man and Glc residues elicited better survival. Strikingly, we observed that tumor cells, that harbored distinct glyco-profiles, also differentially impacted cytokine production by DC subsets. GlcNAc exhibited a negative influence on cDC2s, whereas Fuc and Gal motifs displayed inhibitory impacts on cDC1s and pDCs. Interestingly, blocking specific glycans on melanoma tumor cells restored potent DCs' functionality. The tumor glyco-code was also linked to the nature and density of the immune infiltrate.Such understanding uncovers critical and distinct impact of each DC subset on clinical outcomes, and unveils fine-tuning of interconnections between DCs in melanoma. Our study also revealed the impact of glycan patterns on immunity and suggests that melanoma may manipulate CLR pathways to hijack DC subsets and escape from immune control. Glycans/lectins interactions arise as promising immune checkpoints to rescue DCs from melanoma's hijacking in order to reshape potent antitumor immunity and inhibit immunosuppressive circuits triggered by aberrant tumor glycosylation

Subversion des sous-types de cellules dendritiques BDCA1+ cDC2s, BDCA2+ pDCs et BDCA3+ cDC1s en contexte de mélanome : implication de l'axe glycan / récepteur lectine de type C

Subversion of immunity is a hallmark of cancer development. Dendritic cells (DCs) are strategic immune cells, composed of specialized subsets (cDC1s, cDC2s, pDCs), that initiate and shape anti-tumor immune responses, but tumor cells exploit their plasticity to subvert their functions and escape immune control. The bases for such DCs' hijacking are yet to be fully elucidated. Tumor cells harbor unusual glycosylation patterns of surface glycoproteins and glycolipids that can be sensed through C-type lectin receptors (CLR), expressed on DCs, and subsequently trigger and orientate antitumor immunity. Yet, the pathophysiological role of DCs subsets and the status of their CLR machinery remained largely unknown in the context of melanoma. Furthermore, the global tumor glyco-code and its impact on immunity remains to be elucidated.We investigated the phenotypic (co-stimulatory molecules, CLR patterns) and functional features of circulating and tumor-infiltrating BDCA1+ cDC2s, BDCA2+ pDCs, BDCA3+ cDC1s of melanoma patients, assessed their clinical impact, and further depicted the correlations between CLR expression profiles and other DCs' features. Moreover, to decrypt the potential link between aberrant glycosylation patterns and immune evasion in melanoma, we investigated the melanoma tumor glyco-code through the GLYcoPROFILE™ methodology (lectin arrays), and depicted its impact on patients' clinical outcome and DC subsets' functionality.DC subsets exhibited perturbed frequencies in the circulation and actively infiltrated the tumor site, while harboring a higher activation status. Whereas cDC2s and pDCs displayed an altered functionality in response to TLR-triggering, circulating and tumor-infiltrating cDC1s preserved potent competences associated with improved prognosis. We further highlighted that the CLR repertoire of circulating and tumor-infiltrating cDC1s, cDC2s, and pDCs was strongly perturbed in melanoma patients, with modulation of DCIR, CLEC-12α and NKp44 on circulating DCs, and perturbation of Dectin-1, CD206, DEC205, DC-SIGN and CLEC-9α on tumor-infiltrating DCs. Notably, higher frequencies of circulating cDC1s and higher proportions of DCIR-, DEC205-, CLEC-12α-expressing cDCs were linked with a better clinical outcome of melanoma patients. On the other hand, elevated proportions of CD206-, Dectin1-expressing cDCs and NKp44-expressing pDCs were associated with a poor outcome. Furthermore, when studying the melanoma glyco-code, specific glycan patterns correlated with clinical outcome of melanoma patients, GlcNAc, NeuAc, TF-Ag and Fuc motifs being associated with poor outcome, whereas Man and Glc residues elicited better survival. Strikingly, we observed that tumor cells, that harbored distinct glyco-profiles, also differentially impacted cytokine production by DC subsets. GlcNAc exhibited a negative influence on cDC2s, whereas Fuc and Gal motifs displayed inhibitory impacts on cDC1s and pDCs. Interestingly, blocking specific glycans on melanoma tumor cells restored potent DCs' functionality. The tumor glyco-code was also linked to the nature and density of the immune infiltrate.Such understanding uncovers critical and distinct impact of each DC subset on clinical outcomes, and unveils fine-tuning of interconnections between DCs in melanoma. Our study also revealed the impact of glycan patterns on immunity and suggests that melanoma may manipulate CLR pathways to hijack DC subsets and escape from immune control. Glycans/lectins interactions arise as promising immune checkpoints to rescue DCs from melanoma's hijacking in order to reshape potent antitumor immunity and inhibit immunosuppressive circuits triggered by aberrant tumor glycosylation.La subversion de la réponse immunitaire par les tumeurs est une étape cruciale pour leur développement. Les cellules dendritiques (DCs), composées des sous-types BDCA1+ cDC2s, BDCA2+ pDCs et BDCA3+ cDC1s, sont essentielles pour l'initiation et l'orientation des réponses immunes anti-tumorales. Cependant, les cellules tumorales subvertissent leurs fonctions, en exploitant leur plasticité, afin d'échapper au contrôle du système immunitaire. L'origine de cette subversion n'a pas encore été totalement élucidée. Les cellules tumorales possèdent à leur surface une glycosylation aberrante des glycoprotéines et glycolipides qui peut être reconnue par les récepteurs lectine de type C (CLR), exprimés par les DCs et cruciaux pour déclencher et orienter les réponses subséquentes. Néanmoins, le rôle pathophysiologique des sous-types de DCs et leurs profils d'expression des CLRs reste largement inconnu en contexte du mélanome. De plus, le glyco-code tumoral et son impact sur l'immunité est encore à élucider.Nous avons examiné, chez des patients atteints d'un mélanome, les caractéristiques phénotypiques (molécules de co-stimulation, CLRs) et fonctionnelles des sous-types de DCs circulantes ou infiltrant la tumeur. Nous avons évalué l'impact clinique de ces observations et les corrélations entre les profils d'expression des CLRs et les caractéristiques des DCs. Afin de décrypter le lien potentiel entre les profils aberrants de glycosylation et l'évasion immunitaire en contexte du mélanome, nous avons étudié le glyco-code des cellules tumorales dérivées de patients grâce à la technologie GLYcoPROFILE™ et étudié sa relevance clinique et son impact sur la fonctionnalité des DCs.Les sous-types de DCs présentent des fréquences perturbées dans le sang et infiltrent la tumeur. Les cDC2s et pDCs exhibent une fonctionnalité altérée en réponse à la stimulation TLR, tandis que les cDC1s (circulantes ou infiltrant la tumeur) préservent leur compétences anti-tumorales et sont associées avec un meilleur pronostic des patients. De plus, nous avons pu mettre en évidence une forte modulation du répertoire des CLRs des sous-types de DCs circulantes ou infiltrant la tumeur dans les patients atteint d'un mélanome. En effet, les expressions de DCIR, Clec12α et NKp44 sont modulées sur les DCs circulantes, tandis que les niveaux de Dectin-1, CD206, DEC205, DC-SIGN et Clec9α sont altérés dans les DCs infiltrant la tumeur. De façon intéressante, des fortes proportions de cDC1s circulantes et de cDCs exprimant DCIR, DEC205 et Clec12α sont corrélées avec une meilleure survie des patients. D'autre part, de fortes proportions de cDCs CD206+ ou Dectin1+ et pDCs NKp44+ étaient associées avec un mauvais impact clinique. De plus, l'étude du glyco-code des cellules tumorales a révélé que les motifs GlcNAc, NeuAc, TF-Ag et Fuc sont associés avec un mauvais pronostic clinique, tandis que Man et Glc ont entraîné une meilleure survie. Nous avons aussi pu observer que les cellules tumorales ayant un glyco-code distinct impactaient différemment la production de cytokines par les sous-types de DCs. Une forte expression de GlcNAc avait une influence négative sur les cDC2s, tandis que de forts niveaux des motifs Fuc et Gal impactaient négativement les cDC1s et pDCs. Le blocage spécifique de certains glycans a permis de restaurer la fonctionnalité des DCs. Le glyco-code tumoral était aussi lié avec la nature et densité de l'infiltrat.Cette étude révèle l'impact critique des sous-types de DCs sur le pronostic clinique et l'effet des glycans sur l'immunité, et suggère que le mélanome peut manipuler les CLRs pour détourner les DCs et échapper au contrôle immunitaire. Les interactions glycans-CLR émergent comme de nouveaux points de contrôle immunitaires, en contexte de mélanome, permettant de restaurer la subversion des DCs afin de rétablir une réponse anti-tumorale efficace et d'inhiber les voies immunosuppressives déclenchées par la glycosylation aberrante des tumeurs

Diversification of circulating and tumor‐infiltrating plasmacytoid DCs towards the P3 (CD80 + PDL1 − )‐pDC subset negatively correlated with clinical outcomes in melanoma patients

International audienc

The melanoma tumor glyco-code impacts human dendritic cells’ functionality and dictates clinical outcomes

International audienceSubversion of immunity is a hallmark of cancer development. Dendritic cells (DCs) are strategic immune cells triggering anti-tumor immune responses, but tumor cells exploit their versatility to subvert their functions. Tumor cells harbor unusual glycosylation patterns, which can be sensed through glycan-binding receptors (lectins) expressed by immune cells that are crucial for DCs to shape and orientate antitumor immunity. Yet, the global tumor glyco-code and its impact on immunity has not been explored in melanoma. To decrypt the potential link between aberrant glycosylation patterns and immune evasion in melanoma, we investigated the melanoma tumor glyco-code through the GLYcoPROFILE™ methodology (lectin arrays), and depicted its impact on patients’ clinical outcome and DC subsets’ functionality. Specific glycan patterns correlated with clinical outcome of melanoma patients, GlcNAc, NeuAc, TF-Ag and Fuc motifs being associated with poor outcome, whereas Man and Glc residues elicited better survival. Strikingly, tumor cells differentially impacting cytokine production by DCs harbored distinct glyco-profiles. GlcNAc exhibited a negative influence on cDC2s, whereas Fuc and Gal displayed inhibitory impacts on cDC1s and pDCs. We further identified potential booster glycans for cDC1s and pDCs. Targeting specific glycans on melanoma tumor cells restored DCs’ functionality. The tumor glyco-code was also linked to the nature of the immune infiltrate. This study unveils the impact of melanoma glycan patterns on immunity, and paves the way for innovative therapeutic options. Glycans/lectins interactions arise as promising immune checkpoints to rescue DCs from tumor’ hijacking to reshape antitumor immunity and inhibit immunosuppressive circuits triggered by aberrant tumor glycosylation

BDCA1 + cDC2s, BDCA2 + pDCs and BDCA3 + cDC1s reveal distinct pathophysiologic features and impact on clinical outcomes in melanoma patients

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Melanoma tumour‐derived glycans hijack dendritic cell subsets through C ‐type lectin receptor binding

International audienceAbstract Dendritic cell (DC) subsets play a crucial role in shaping anti‐tumour immunity. Cancer escapes from the control immune system by hijacking DC functions. Yet, bases for such subversion are only partially understood. Tumour cells display aberrant glycan motifs on surface glycoproteins and glycolipids. Such carbohydrate patterns can be sensed by DCs through C‐type lectin receptors (CLRs) that are critical to shape and orientate immune responses. We recently demonstrated that melanoma tumour cells harboured an aberrant ‘glyco‐code,’ and that circulating and tumour‐infiltrating DCs from melanoma patients displayed major perturbations in their CLR profiles. To decipher whether melanoma, through aberrant glycan patterns, may exploit CLR pathways to mislead DCs and evade immune control, we explored the impact of glycan motifs aberrantly found in melanoma (neoglycoproteins [NeoGP] functionalised with Gal, Man, GalNAc, s‐Tn, fucose [Fuc] and GlcNAc residues) on features of human DC subsets (cDC2s, cDC1s and pDCs). We examined the ability of glycans to bind to purified DCs, and assessed their impact on DC basal properties and functional features using flow cytometry, confocal microscopy and multiplex secreted protein analysis. DC subsets differentially bound and internalised NeoGP depending on the nature of the glycan. Strikingly, Fuc directly remodelled the expression of activation markers and immune checkpoints, as well as the cytokine/chemokine secretion profile of DC subsets. NeoGP interfered with Toll like receptor (TLR)‐signalling and pre‐conditioned DCs to exhibit an altered response to subsequent TLR stimulation, dampening antitumor mediators while triggering pro‐tumoral factors. We further demonstrated that DC subsets can bind NeoGP through CLRs, and identified GalNAc/MGL and s‐Tn/ C‐type lectin‐like receptor 2 (CLEC2) as potential candidates. Moreover, DC dysfunction induced by tumour‐associated carbohydrate molecules may be reversed by interfering with the glycan/CLR axis. These findings revealed the glycan/CLR axis as a promising checkpoint to exploit in order to reshape potent antitumor immunity while impeding immunosuppressive pathways triggered by aberrant tumour glycosylation patterns. This may rescue DCs from tumour hijacking and improve clinical success in cancer patients

Dysfunctional BTN3A together with deregulated immune checkpoints and type I/II IFN dictate defective interplay between pDCs and γδ T cells in melanoma patients, which impacts clinical outcomes

International audienceObjectives: pDCs and γδ T cells emerge as potent immune players participating in the pathophysiology of cancers, yet still remaining enigmatic while harbouring a promising potential for clinical translations. Despite strategic and closed missions, crosstalk between pDCs and γδ T cells has not been deciphered yet in cancers, especially in melanoma where the long-term control of the tumor still remains a challenge.Methods: This prompted us to explore the interplay between pDCs and γδ T cells in the context of melanoma, investigating the reciprocal features of pDCs or γδ T cells, the underlying molecular mechanisms and its impact on clinical outcomes.Results: TLRL-activated pDCs from the blood and tumor infiltrate of melanoma patients displayed an impaired ability to activate, to modulate immune checkpoints and trigger the functionality of γδ T cells. Conversely, γδ T cells from the blood or tumor infiltrate of melanoma patients activated by PAg were defective in triggering pDCs' activation and modulation of immune checkpoints, and failed to elicit the functionality of pDCs. Reversion of the dysfunctional cross-talks could be achieved by specific cytokine administration and immune checkpoint targeting. Strikingly, we revealed an increased expression of BTN3A on circulating and tumor-infiltrating pDCs and γδ T cells from melanoma patients, but stressed out the potential impairment of this molecule.Conclusion: Our study uncovered that melanoma hijacked the bidirectional interplay between pDCs and γδ T cells to escape from immune control, and revealed BTN3A dysfunction. Such understanding will help harness and synergise the power of these potent immune cells to design new therapeutic approaches exploiting their antitumor potential while counteracting their skewing by tumors to improve patient outcomes

Hepatitis B virus exploits C‐type lectin receptors to hijack cDC1s, cDC2s and pDCs

International audienceC-type lectin receptors (CLRs) are key receptors used by DCs to orchestrate responses to pathogens. During infections, the glycan-lectin interactions shape the virus-host interplay and viruses can subvert the function of CLRs to escape antiviral immunity. Recognition of virus/viral components and uptake by CLRs together with subsequent signalling cascades are crucial in initiating and shaping antiviral immunity, and decisive in the outcome of infection. Yet, the interaction of hepatitis B virus (HBV) with CLRs remains largely unknown. As HBV hijacks DC subsets and viral antigens harbour glycan motifs, we hypothesised that HBV may subvert DCs through CLR binding