As expected, the MHC-class II-restricted CD4+ T cell proliferation was compromised in the Cat-S KO BM-DCs (data not shown), illustrating the involvement of Cathepsin-S in cleaving the invariant chain of the MHC-class II molecule (Nakagawa et al

As expected, the MHC-class II-restricted CD4+ T cell proliferation was compromised in the Cat-S KO BM-DCs (data not shown), illustrating the involvement of Cathepsin-S in cleaving the invariant chain of the MHC-class II molecule (Nakagawa et al., 1999). Open in a separate window Figure 5. LeX-modified antigen is cross-presented in a TAP- and Cathepsin-S-independent fashion.To examine whether cross-presentation of OVA-LeX involves TAP or Cathepsin-S (A) TAP1 KO and (B) Cat-S KO BM-DCs and WT BM-DCs were pulsed with OVA-LeX or native OVA and co-cultured with OT-I T cells for 3 days. nature and strength of immune responses and should be considered for optimizing current vaccination strategies. DOI: http://dx.doi.org/10.7554/eLife.11765.001 with either OVA-LeX or native OVA mixed with anti-CD40 using a prime-boost protocol. Spleens were analyzed by flow cytometry to determine the frequency of (C) H2-Kb/SIINFEKL-tetramer-binding CD8+ T cells and IFN- or TNF production by activated CD8+ T cells was determined by intracellular staining after OVA-specific re-stimulation ex vivo. Dots represent individual mice (n=4C5 mice/group; **p<0.01). Bars indicate median of each group. Graphs shown are representative of two independent experiments. (D) C57BL/6 and MGL1 KO mice were prime-boosted with either OVA-LeX or native OVA mixed with anti-CD40. Frequencies of IFN- and TNF-double-producing CD8+ T BJE6-106 cells were determined by intracellular staining after OVA-specific re-stimulation of splenocytes ex vivo. Dots represent individual mice (n=4C5 mice/group; *p<0.05 ***p<0.001). Bars indicate median of each group. Data are representative of 2 independent experiments. DOI: http://dx.doi.org/10.7554/eLife.11765.005 Figure 2figure supplement 1. Open in a separate window Representative flow cytometry plots of (A) IFN- and (B) TNF- producing CD8+ T cells in spleens of C57BL/6 mice that were immunized with either OVA-LeX or native OVA mixed with anti-CD40 using a prime-boost protocol; numbers above the gates designate the percentage of IFN-+ or TNF+ CD8+ T cells.DOI: http://dx.doi.org/10.7554/eLife.11765.006 Figure 2figure supplement 2. Open in a separate window C57BL/6 and MGL1 KO mice were prime-boosted with either OVA-LeX or native OVA mixed with anti-CD40.Frequencies of IFN- and TNF-double-producing CD8+ T cells were determined by intracellular staining after re-stimulation of splenocytes ex vivo. Representative facs plots of indicated mice are shown; numbers designate the percentage BJE6-106 of IFN- and TNF-double positive CD8+ T cells. DOI: http://dx.doi.org/10.7554/eLife.11765.007 OVA-LeX induces Th1 skewing of naive CD4+ T cells Since we observed that LeX-modified Mouse monoclonal to NSE. Enolase is a glycolytic enzyme catalyzing the reaction pathway between 2 phospho glycerate and phosphoenol pyruvate. In mammals, enolase molecules are dimers composed of three distinct subunits ,alpha, beta and gamma). The alpha subunit is expressed in most tissues and the beta subunit only in muscle. The gamma subunit is expressed primarily in neurons, in normal and in neoplastic neuroendocrine cells. NSE ,neuron specific enolase) is found in elevated concentrations in plasma in certain neoplasias. These include pediatric neuroblastoma and small cell lung cancer. Coexpression of NSE and chromogranin A is common in neuroendocrine neoplasms. OVA increased priming of antigen-specific CD8+ T cells we examined whether this also enhanced antigen-presentation to CD4+ T cells. Both OVA-LeX-loaded and native OVA-loaded spDCs induced CD4+ OT-II T cell proliferation to a similar extent (Figure 3A), illustrating that the altered antigen uptake mediated by LeX did not affect loading on MHC class II molecules. Similar results were obtained using BM-DCs (Figure 3A). Although we did not observe any differential effect of LeX on CD4+ T cell expansion, neoglycosylation of antigens could induce signaling via CLRs and herewith potentially influence Th cell differentiation (Gringhuis et al., 2014). We therefore investigated whether BJE6-106 OVA-LeX affected the differentiation of naive CD4+ T cells. Hereto BM-DCs and spDCs of C57BL/6 mice were pulsed with OVA-LeX and subsequently co-cultured with naive CD4+CD62Lhi OT-II cells. Co-cultures containing OVA-LeX loaded BM-DCs or spDCs contained significantly more IFN–producing T cells than those containing OVA-loaded DCs (Figure 3B). Neither induction of IL-4- nor IL-17A-producing CD4+ T cells was observed (Figure 3B, upper and middle panel and data not shown). In addition, induction of Foxp3+ T cells was not detected (data not shown). To exclude that the Th1 skewing by OVA-LeX loaded DCs was attributed to the more Th1 prone status of C57BL/6 (Gervais et al., 1984), we also performed the Th-differentiation assay with cells derived from Th2 prone BALB/c mice (Hsieh et al., 1995). We observed that naive OVA-specific CD4+ T cells from DO11.10 Tg mice that were stimulated with OVA-loaded BM-DCs differentiated into IL-4 secreting T cells (Figure 3B, lower panels). However, the generation of IL-4-producing T cells was not influenced by loading DCs with OVA-LeX as these cultures contained comparable percentages of IL-4-producing DO11.10?T cells. Using these Th2-prone T cells, OVA-LeX-pulsed DCs still induced considerably more IFN–producing CD4+ T cells than native OVA-pulsed DCs (Figure 3B, lower panel). Since this assay takes three days longer than the antigen-presentation assay, it is possible that the higher frequency of IFN–producing CD4+ T cells is due to increased division of OVA-specific CD4+ T cells. However we found that the amount of proliferation of OVA-specific CD4+ T cells induced by stimulation with OVA-LeX-loaded DCs after 6 days is similar to that induced by OVA-loaded DCs (Figure 3figure supplement 1). The augmented induction of CD4+ Th1 cells was also observed in vivo as revealed from the higher frequencies of IFN–producing OVA-specific CD4+ T cells in the spleens of OVA-LeX immunized mice than in mice immunized with native OVA (Figure 3C, Figure 3figure supplement 2). These data indicate that the increased numbers of Th1 cells induced by.