IFNDCs (5 103) were loaded with 1 g/ml recombinant fusion proteins of PSA, pooled peptides (10 M), or none overnight. cells through intercellular relationships and soluble factors (Banchereau and Steinman, 1998; Rissoan et al., 1999; Akira et al., 2001; Soares et al., 2007), resulting in different quality and quantity of sponsor immune reactions. In addition, different subsets of DCs display common and unique biological functions for controlling sponsor immune reactions (Caux et al., 1996; Maldonado-Lpez et al., 1999; Pulendran et al., 1999; Banchereau et al., 2000; Shortman and Liu, 2002; Dudziak et al., 2007; Soares et al., 2007; Klechevsky et al., 2008). DCs communicate pattern acknowledgement receptors (Figdor et al., 2002; Geijtenbeek et al., 2004; Brown, 2006), most notably displayed by Toll-like receptors (Akira et al., 2001) and lectinlike receptors (LLRs; Figdor et al., 2002; Geijtenbeek et al., 2004; Brown, 2006; Caparrs et al., 2006). Ligation of TLRs results in the activation of DCs, followed by cytokine and chemokine secretion that contribute to the outcomes of sponsor immune reactions. LLRs operate as constituents of the powerful antigen capture and uptake system (Delneste et al., 2002; Figdor et al., 2002; Geijtenbeek et al., 2004; Brown, 2006; Geijtenbeek and Gringhuis, 2009). Recent compelling evidence also indicates that individual LLRs indicated on DCs might possess unique functions (Delneste et al., 2002; Figdor et al., 2002; Brown, 2006; Geijtenbeek and Gringhuis, 2009), that can contribute to shaping the quality and quantity of sponsor immune reactions. For example, lectinlike oxidized-LDL receptor (LOX-1), Dectin-1, and DC-specific ICAM-3Cgrabbing nonintegrin (DC-SIGN) are capable of delivering intracellular signals, either by themselves or by combination with TLRs, that activate DCs and may result in modified T cell reactions (Figdor et al., 2002; Delneste et al., 2002; Geijtenbeek et al., 2004; Smits et al., 2005; Brown, 2006; Caparrs et al., 2006; Dillon et al., 2006; Geijtenbeek and Gringhuis, 2009; Geurtsen et al., 2009). Particular features of LLRsantigen capture, uptake, and signaling capacityplace them as important immune receptors that could determine the outcomes of sponsor immune responses. Indeed, DCs triggered via Dectin-1 result in polarized Th17 CD4+ T cell reactions (LeibundGut-Landmann et al., 2007; Gringhuis et al., 2009). It was also reported that signals via Dectin-1 induce IL-10 in DCs (Rogers et al., 2005; Ni et al., 2010), and activation of DCs via Dectin-1 and TLR2 results in regulatory T cell reactions (Dillon et al., 2006). DC-SIGN binding by different pathogens can lead to promotion of Th2 reactions (Bergman et al., 2004; Caparrs et al., 2006; Geurtsen et al., 2009) and the induction of regulatory T cell differentiation (Smits et al., 2005; Geijtenbeek and Gringhuis, 2009). DC-asialoglycoprotein receptor (DC-ASGPR) is definitely a scavenger receptor transporting an immunoreceptor tyrosine-based activation motiflike motif (Valladeau et al., 2001), but the biological function of DC-ASGPR indicated on DCs has not been characterized. In this study, we demonstrate for the first time that DC-ASGPR has a novel function for generating antigen-specific IL-10Cgenerating suppressive CD4+ T cells in vitro. Furthermore, antigens fused to antiCDC-ASGPR antibody can generate IL-10Cgenerating antigen-specific T cells in macaques. This study provides a novel strategy for the establishment of antigen-specific IL-10Cgenerating regulatory T cells in vivotest. (B) CD4+ T cells were restimulated with indicated peptides and stained for intracellular IL-10. HA1280-296 is definitely a negative control. Peptides tested in B had been selected in Fig. S2 (F and G). Four self-employed experiments showed related results. (C) Naive CD4+ T cells (1C2 105) were co-cultured with IFNDCs (5 103) loaded with 1 g/ml recombinant fusion proteins for 7 d. CD4+ T cells were restimulated with peptides indicated for 48 h. IL-10 and IFN- levels in culture supernatants were assessed. Error bars represent mean SEM of triplicate assay. Three impartial experiments with 59 PSA-derived peptides showed similar results. (D) Frequency of PSA-specific IL-10Cproducing CD4+ T cells elicited by recombinant fusion proteins. Four independent experiments showed similar results. (E) Experiments in C were performed with blood mDCs (Lin?HLA-DR+CD11c+CD123?). (F) Experiments in D were performed with blood mDCs. In both E and F, two independent experiments showed similar results. (G) Expression levels of Foxp3, PD-1, and CTLA-4 on HA1250-266-specific (left) and PSA30-44-specific CD4+ T cells producing IL-10 (right). (H) After 7 d of co-culture of purified naive CD4+ T cells and DCs loaded with either -LOX-1-PSA or -DC-ASGPR-PSA, CD4+ T cells were stained for intracellular IFN- and IL-10 during restimulation with 50 ng/ml PMA.S4 demonstrates that both HA1 and PSA delivered to DCs via DC-ASGPR results in enhanced HA1- and PSA-specific Ezutromid IL-10Cproducing CD4+ T cell responses. As immune controllers, DCs can deliver differential signals to other immune cells through intercellular interactions and soluble factors (Banchereau and Steinman, 1998; Rissoan et al., 1999; Akira et al., 2001; Soares et al., 2007), resulting in different quality and quantity of host immune responses. In addition, different subsets of DCs display common and unique biological functions for controlling host immune responses (Caux et al., 1996; Maldonado-Lpez et al., 1999; Pulendran et al., 1999; Banchereau et al., 2000; Shortman and Liu, 2002; Dudziak et al., 2007; Soares et al., 2007; Klechevsky et al., 2008). DCs express pattern recognition receptors (Figdor et al., 2002; Geijtenbeek et al., 2004; Brown, 2006), most notably represented by Toll-like receptors (Akira et al., 2001) and lectinlike receptors (LLRs; Figdor et al., 2002; Geijtenbeek et al., 2004; Brown, 2006; Caparrs et al., 2006). Ligation of TLRs results in the activation of DCs, followed by cytokine and chemokine secretion that contribute to the outcomes of host immune responses. LLRs operate as constituents of the powerful antigen capture and uptake system (Delneste et al., 2002; Figdor et al., 2002; Geijtenbeek et al., 2004; Brown, 2006; Geijtenbeek and Gringhuis, 2009). Recent compelling evidence also indicates that individual LLRs expressed on DCs might possess unique functions (Delneste et al., 2002; Figdor et al., 2002; Brown, 2006; Geijtenbeek and Gringhuis, 2009), that can contribute to shaping the quality and quantity of host immune responses. For example, lectinlike oxidized-LDL receptor (LOX-1), Dectin-1, and DC-specific ICAM-3Cgrabbing nonintegrin (DC-SIGN) are capable of delivering intracellular signals, either by themselves or by combination with TLRs, that activate DCs and can result in altered T cell responses (Figdor et al., 2002; Delneste et al., 2002; Geijtenbeek et al., 2004; Smits et al., 2005; Brown, 2006; Caparrs et al., 2006; Dillon et al., 2006; Geijtenbeek and Gringhuis, 2009; Geurtsen et al., 2009). Certain features of LLRsantigen capture, uptake, and signaling capacityplace them as key immune receptors that could determine the outcomes of host immune responses. Indeed, DCs activated via Dectin-1 result in polarized Th17 CD4+ T cell responses (LeibundGut-Landmann et al., 2007; Gringhuis et al., 2009). It was also reported that signals via Dectin-1 induce IL-10 in DCs (Rogers et al., 2005; Ni et al., 2010), and activation of DCs via Dectin-1 and TLR2 results in regulatory T cell responses (Dillon et al., 2006). DC-SIGN binding by different pathogens can lead to promotion of Th2 responses (Bergman et al., 2004; Caparrs et al., 2006; Geurtsen et al., 2009) and the induction of regulatory T cell differentiation (Smits et al., 2005; Geijtenbeek and Gringhuis, 2009). DC-asialoglycoprotein receptor (DC-ASGPR) is usually a scavenger receptor carrying an immunoreceptor tyrosine-based activation motiflike motif (Valladeau et al., 2001), but the biological function of DC-ASGPR expressed on DCs has not been characterized. In this study, we demonstrate for the first time that DC-ASGPR has a novel function for generating antigen-specific IL-10Cproducing suppressive CD4+ T cells in vitro. Furthermore, antigens fused to antiCDC-ASGPR antibody can generate IL-10Cproducing antigen-specific T cells in macaques. This study provides a novel strategy for the establishment of antigen-specific IL-10Cproducing regulatory T cells in vivotest. (B) CD4+ T cells were restimulated with indicated peptides and stained for intracellular IL-10. HA1280-296 is usually a negative control. Peptides tested in B had been selected in Fig. S2 (F and G). Four impartial experiments showed comparable results. (C) Naive CD4+ T cells (1C2 105) were co-cultured with IFNDCs (5 103) loaded with 1 g/ml recombinant fusion proteins for 7 d. CD4+ T cells were restimulated with peptides indicated for 48 h. IL-10 and IFN- levels in culture supernatants were assessed. Error bars represent mean SEM of triplicate assay. Three Ezutromid impartial experiments with 59 PSA-derived peptides showed similar results. (D) Frequency of PSA-specific IL-10Cproducing CD4+ T cells elicited by recombinant fusion proteins. Four independent experiments showed similar results. (E) Experiments in C were performed with blood mDCs (Lin?HLA-DR+CD11c+CD123?). (F) Experiments in Ezutromid D were performed with blood Pax1 mDCs. In both E and F, two impartial experiments showed comparable results. (G) Expression levels of Foxp3, PD-1, and CTLA-4 on HA1250-266-specific (left) and PSA30-44-specific CD4+ T cells producing IL-10 (right). (H) After 7 d of co-culture of purified naive CD4+ T cells and DCs loaded with either -LOX-1-PSA or -DC-ASGPR-PSA, CD4+ T cells were stained for intracellular IFN- and IL-10 during restimulation with 50 ng/ml.