Elucidating Molecular Mechanisms of Cross-Presentation

The immune system regulates many processes vital to homeostasis. Antigen cross-presentation, an immune process specific to dendritic cells (DCs), critically contributes to maintaining homeostasis by regulating immune tolerance, antiviral activity, and antitumor responses. Through cross-presentation, extracellular antigen is processed and presented by MHC I on DCs to CD8 T cells. Despite cross-presentation’s crucial role in mediating immune responses, its molecular regulation remains poorly defined. A potential breakthrough came when, using an in vitro shRNA-mediated knockdown (KD) approach, a group identified Sec22b as a central regulatory molecule of the pathway¹. They demonstrated that Sec22b mediates recruitment of proteins necessary for MHC I-antigen loading from the endoplasmic reticulum (ER)-Golgi intermediate compartment (ERGIC) to the antigen-containing phagosome, promoting cross-presentation. Thus, we tested the hypothesis that Sec22b mediates cross-presentation in vivo, in the context of an intact immune system under physiological conditions. We generated DC-specific Sec22b knockout (KO) mice (CD11c^Cre Sec22b^fl/fl) from Sec22b-conditional gene trapped founder mice. These mice develop normally and have an intact immune system. KO DCs from spleen and bone marrow (BM) have Cre-mediated excision at the Sec22b locus, verified by PCR, and reduction of Sec22b production, verified by Western Blot. KO DCs from spleen and BM express activation markers in response to TLR and NLR stimulation at comparable levels to Cre^– Sec22b^fl/fl (FL) mice. By adoptively transferring ovalbumin (OVA)-specific (OT-I) T cells into these mice, then injecting with soluble OVA i.p., we measured OT-I T cell proliferation as a readout of cross-presentation. To our surprise, we saw no difference in the ability of KO versus FL mice to cross-present OVA (p>0.9). This observation was verified with in vitro assays with KO DCs from BM and spleen cross-presenting soluble OVA (0-3 mg/mL). We obtained similar findings using bead-bound, insoluble OVA. From this, we concluded Sec22b is not necessary for cross-presentation, invalidating our hypothesis. We were, however, able to reduce cross-presentation by shRNA-mediated KD of Sec22b (p<0.05), reproducing published observations.  This discrepancy in observations was not due to functional compensation in KO BMDCs by Sec22b homologs, Sec22a or Sec22c, which we determined using qPCR. Intriguingly, when we treated KO BMDCs with the Sec22b-targeting shRNA, we again observed a reduction in cross-presentation (p<0.05). The reduction was comparable to that found in Sec22b-targeting shRNA-treated FL and WT BMDCs. Taken together, our data (a) demonstrate that Sec22b is not necessary for cross-presentation, (b) suggest the existence of a novel critical mediator of cross-presentation that is also targeted by the shRNA sequence used and (c) caution against extrapolating mechanisms or phenotypes based on KD studies alone.

1. Cebrian, I. et al. Sec22b regulates phagosomal maturation and antigen crosspresentation by dendritic cells. Cell 147, 1355-1368 (2011).