As the mannose receptor is expected to recognize glycan components of ovalbumin, it was also of relevance to this study to compare cross presentation efficiencies of the non-glycosylated E. coli-derived ovalbumin with that of glycosylated ovalbumin derived from a natural source, which was undertaken. This possibility is further supported by the analyses shown in Figure 5, where no significant differences were measurable between crosspresentation efficiencies of ovalbumin derived from E.coli or chicken eggs, which are expected to be non-glycosylated and glycosylated respectively. Altogether, the studies described in Figures 2, 3, and 5 taken together with previous studies indicate that multiple uptake pathways can contribute to the crosspresentation of ovalbumin. Regardless of the precise uptake pathway, although a specific cross-presentation advantage that results from the calreticulin fusion is not apparent. Thus, if calreticulin-specific receptors contribute to increased uptake of the OVA-CRT fusion in vitro or in vivo, such uptake does not INCB18424 result in increased cross-presentation. Taken together, our studies suggest that the immunogenic properties of calreticulin purified from tumor cells must result from co-purification and subsequent cross-presentation of one or more tumor-derived antigens, rather than a calreticulindependent influence on the cross-presentation pathway per se. By binding to antigen within its substrate binding site, calreticulin could protect antigen from complete proteolytic degradation, thus preserving antigen in a form that is competent for subsequent cross-presentation. General uptake pathways for soluble antigen may be operative during the cross-presentation of calreticulinantigen complexes, and it is possible that calreticulin binding can confer a kinetic advantage for cross-presentation over complete degradation, at least for some antigens. The latter mechanism might explain previous findings of the potentiating activity of calreticulin during cross-presentation of elongated peptides. It remains possible that the soluble OVA-CRT construct may not have been able to bind calreticulin-specific receptors with a high enough avidity to impact the cross-presentation of OVA. OVA may have masked the calreticulin-receptor binding site on calreticulin. To address this issue, calreticulin and OVA or OVA alone were conjugated to iron oxide beads and cross-presentation efficiencies were assessed. OVA and calreticulin are not fused in this system. Thus, calreticulin-receptor interactions should not be inhibited. We show that calreticulin was not able to enhance the cross-presentation of the bead-associated OVA compared to beads with OVA alone, both in vitro and in vivo. Calreticulin has been reported to be an “eat me” signal on the surface of apoptotic cells. The findings of Figure 4 suggest that calreticulin does not work independently in a phagocytic context, but rather might work in conjunction with other “eat me” signals such as phosphatidylserine. Hence, calreticulin in isolation is not sufficient to enhance cross-presentation of a particulate antigen. However, it is also possible that phagocytic uptake of the iron oxide beads is intrinsically high, even in the absence of calreticulin. In summary, we have examined whether calreticulin can influence CD8 T cell proliferation against peptide, soluble and bead-associated antigen. We show that ovalbumin is crosspresented with similar efficiency when delivered alone compared to delivery with calreticulin as a soluble fusion, or co-conjugated on beads.