If it ain’t broke, break it: facilitating antigen cross-presentation
Authors: Pablo Rodríguez-Silvestre, Patrycja Kozik
Journal: Trends in Molecular Medicine (2022)

Recently, Bikorimana presented a new vaccine formulation, AccumTM, capable of eliciting efficient T cell-mediated immune responses and controlling tumour growth. AccumTM is designed to facilitate cross-presentation by breaking through endo/lysosomal membranes and enhancing delivery of antigens into the cytosol of dendritic cells (DCs). Recently, Bikorimana presented a new vaccine formulation, AccumTM, capable of eliciting efficient T cell-mediated immune responses and controlling tumour growth. AccumTM is designed to facilitate cross-presentation by breaking through endo/lysosomal membranes and enhancing delivery of antigens into the cytosol of dendritic cells (DCs). DCs appeared on the radar of immunotherapy researchers shortly after their discovery in the 1970s by Ralph Steinman – himself not only a strong advocate for DC-based therapies but also a recipient thereof. The key role of DCs in anticancer immunity is largely ascribed to their superior ability to present tumour-derived antigens to CD8+ T cells and to drive expansion of tumour-specific cytotoxic lymphocytes (CTLs) [1.Theisen D.J. et al.WDFY4 is required for cross-presentation in response to viral and tumor antigens.Science. 2018; 362: 694-699Crossref PubMed Scopus (136) Google Scholar]. While this nonredundant role of DCs in the initiation of antitumour immune responses has now been firmly established, disappointingly, progress with DC-based or DC-targeting vaccines has lagged behind. Efforts to therapeutically boost DC activity can be broadly divided into strategies designed to facilitate presentation of tumour-derived antigens and strategies aimed at promoting DC maturation/abundance by providing appropriate adjuvants and cytokines. This is exemplified by the first DC-based vaccine, Sipuleucel-T, approved by the FDA for treatment of advanced prostate cancer in 2010 [2.Kantoff P.W. et al.Sipuleucel-T immunotherapy for castration-resistant prostate cancer.New Engl. J. Med. 2010; 363: 411-422Crossref PubMed Scopus (4127) Google Scholar]. Sipuleucel-T consists of DCs pulsed ex vivo with a fusion protein of prostate acid phosphatase (an antigen often presented by prostate cancer cells) and GM-CSF (a cytokine driving DC maturation). Although Sipuleucel-T has shown limited clinical benefits to date, better understanding of the functional differences between the different DC subsets and of the immunosuppressive mechanisms in the tumour microenvironment have revived efforts to therapeutically boost DC functions [3.Bhardwaj N. et al.Flt3 ligand augments immune responses to anti-DEC-205-NY-ESO-1 vaccine through expansion of dendritic cell subsets.Nat. Cancer. 2020; 1: 1204-1217Crossref PubMed Scopus (31) Google Scholar]. One of the challenges in designing antigen formulations for cell-based vaccines lies in our incomplete understanding of mechanisms underlying presentation of tumour-associated antigens. DCs are uniquely equipped to acquire antigens from surrounding cells and present them on MHC class I molecules. This process is referred to as cross-presentation and there are currently two models explaining how antigens are processed for loading onto MHC class I by DCs. In the first model ('vacuolar pathway'), the epitopes are generated by lysosomal proteases, similar to the majority of MHC class II-binding peptides. In the second model ('cytosolic pathway') antigens escape from the endo/lysosomal system into the cytosol where they are processed by the proteasome (reviewed in [4.Blander J.M. Regulation of the cell biology of antigen cross-presentation.Annu. Rev. Immunol. 2018; 36: 717-753Crossref PubMed Scopus (78) Google Scholar]). A number of different mechanisms of endosomal escape have been proposed involving either a channel (such as Sec61) or transient loss of endo/lysosomal membrane integrity (e.g., through lipid peroxidation) [5.Gros M. Amigorena S. Regulation of antigen export to the cytosol during cross-presentation.Front. Immunol. 2019; 10: 41Crossref PubMed Scopus (44) Google Scholar]. Which mechanisms are relevant for presentation of tumour-derived antigens, and whether endosomal escape is indeed required for cross-presentation in vivo, are still unclear. Considering that the cytosolic pathway is more likely to generate the same epitopes as those presented on cancer cells, it has received significantly more focus over recent years. In line with this model, protecting antigens from lysosomal degradation or facilitating endosomal escape was shown to enhance cross-presentation both in vitro and in vivo [6.Kozik P. et al.Small molecule enhancers of endosome-to-cytosol import augment anti-tumor immunity.Cell Rep. 2020; 32107905Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar,7.Chatterjee B. et al.Internalization and endosomal degradation of receptor-bound antigens regulate the efficiency of cross presentation by human dendritic cells.Blood. 2012; 120: 2011-2020Crossref PubMed Scopus (143) Google Scholar]. By contrast, interfering with cytosolic delivery of antigens taken up by cross-presentation-competent DCs can be employed by tumours as a means of immune evasion [8.Giampazolias E. et al.Secreted gelsolin inhibits DNGR-1-dependent cross-presentation and cancer immunity.Cell. 2021; 184: 4016-4031.e22Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar]. Improving cytosolic delivery is a challenge faced not only by the DC enthusiasts but also in the drug-delivery field. Taking advantage of this overlap, Bikorimana et al. [9.Bikorimana et al.Promoting antigen escape from dendritic cell endosomes potentiates anti-tumoral immunity.Cell Rep. Med. 2022; (Published online March 15, 2022)https://doi.org/10.1016/j.xcrm.2022.100534Abstract Full Text Full Text PDF PubMed Scopus (4) Google Scholar] set out to boost endosomal escape by employing a protein modification initially developed for delivery of radiolabelled antibodies into the nucleus of the target cells [10.Beaudoin S. et al.ChAcNLS, a novel modification to antibody-conjugates permitting target cell-specific endosomal escape, localization to the nucleus, and enhanced total intracellular accumulation.Mol. Pharmaceut. 2016; 13: 1915-1926Crossref PubMed Scopus (13) Google Scholar]. The modification, referred to as AccumTM, consists of a fusion of cholic acid with a peptide containing a nuclear localization signal (NLS). Cholic acid has been proposed to mediate escape into the cytosol through a mechanism involving ceramide accumulation and consequent disruption of the endo/lysosomal membrane. Here, Bikorimana et al. report that Accum-labelled ovalbumin (Ova) indeed facilitates membrane damage of endo/lysosomal compartments and, once in the cytosol, it undergoes rapid proteasomal degradation. Surprisingly, the NLS-containing peptide with AccumTM is required for the observed enhancement of cross-presentation even though antigen processing takes place in the cytosol rather than in the nucleus. As predicted, facilitating cytosolic escape by using Accum-labelled antigens improved the efficiency of CD8+ T cell priming both in vitro and in vivo. Perhaps more surprisingly, Accum-Ova also boosted the efficiency of CD4+ T cell activation (Figure 1). Accum-Ova-based vaccines also led to increased antibody titres in tumour-bearing mice, and vaccination with allogeneic DCs loaded with Accum-labelled tumour cell lysates enhanced tumour infiltration with DCs but also with natural killer (NK) cells. Overall, the authors elegantly demonstrated the efficacy of both therapeutic and prophylactic vaccination regimens in controlling tumour growth. In one of the multiple models employed, vaccination with syngeneic DCs loaded with Accum-Ova conferred complete protection from multiple challenges with EG.7 T cell lymphoma. Overall, the observation that Accum-based vaccines successfully engage multiple arms of the immune system highlights a longstanding question of what is necessary to efficiently boost CTL-mediated immune responses in vivo. In line with recent studies [11.Ferris S.T. et al.cDC1 prime and are licensed by CD4+ T cells to induce anti-tumour immunity.Nature. 2020; 584: 624-629Crossref PubMed Scopus (130) Google Scholar], the ability of AccumTM to improve both CD8+ and CD4+ T cell responses may partly explain the robust antitumour immunity observed. Further work will be required to establish whether CD4+ T cell activation and other responses triggered by DCs pulsed with Accum-conjugated antigens are all due to the ability of AccumTM to destabilise endo/lysosomal compartments and deliver antigens into the cytosol – or whether other mechanisms contribute. Ultimately, considering that the majority of experimental approaches aimed at promoting antigen delivery into the cytosol involve at least some damage to endocytic compartments, a major challenge will be to establish whether such formulations can be safely used in therapeutics. We would like to thank Marco Laub and Patrycja Krawczyk for comments on the manuscript. The authors are supported by the Medical Research Council (MRF-104-0007-S-RODRI and MC_UP_1201/2). No interests are declared.