CD8α+ dendritic cells (DCs) can acquire preformed peptide–MHC class I complexes and membrane-associated antigens from live tumor cells by trogocytosis rather than by phagocytosing dying cells; incorporation of the acquired preformed peptide–MHC into the DC membrane is termed 'cross-dressing', and this route is a major mechanism of cross-priming of anti-tumor CD8+ T cells after immunization with live tumor cells. The precise molecular signals that make a DC trogocytose a live tumor cell instead of phagocytosing it (beyond the known 'don't-eat-me' signal CD47), whether this route intrinsically avoids the tolerogenic programming induced by phagocytosis of apoptotic corpses, how much cross-dressing contributes relative to classical cross-presentation pathways across tumor models and anatomical sites, and whether it can be exploited therapeutically with live autologous tumor cells as antigen, all remain unresolved.

Background

To initiate a cytotoxic CD8+ T cell response against tumors, rare professional antigen-presenting cells must display tumor-derived peptides on MHC class I molecules to naive T cells. Because tumors are not professional presenters, this display must be accomplished by dendritic cells (DCs), the antigen-presenting cells specialized for activating naive T cells. The classical route is 'cross-presentation': the DC internalizes exogenous tumor protein (for example by phagocytosing dying tumor cells), proteolytically processes it, and loads the resulting peptides onto its own MHC class I molecules; the resulting priming of CD8+ T cells is called 'cross-priming'. In mice, the CD8α+ conventional DC (equivalent to the human cDC1 subset) is the subset most specialized for cross-presentation in vivo.

Two non-classical routes complicate this picture. First, heat shock protein–peptide complexes (notably the chaperone gp96 and its receptor CD91) released or transferred from tumor cells can hand preprocessed peptides to DCs for cross-priming. Second, DCs can acquire intact, preformed peptide–MHC (pMHC) complexes or membrane-associated antigens from other living cells and display them without further processing. One mechanism for this is 'trogocytosis', in which a cell nibbles membrane fragments from a living donor cell by direct contact, usually without killing it; a DC that incorporates acquired pMHC into its own plasma membrane is said to be 'cross-dressed'. Cross-dressing was demonstrated in vitro by Dolan et al. (2006), who showed that DCs pulsed with intact peptide–MHC class I or class II complexes from cell-based cancer vaccines prime CD8+ and CD4+ T cells.

Das Mohapatra et al. (2020) established the in vivo relevance of this route for live-cell immunogenicity. Using tumor cells labeled with the fluorescent dyes CFSE (a cytoplasmic dye) and PKH26 (a dye that inserts into the plasma membrane) injected into mice, they found that live tumor cells were markedly more immunogenic than irradiated or apoptotic tumor cells; that CD8α+ DCs in skin-draining lymph nodes captured membrane-associated antigen from live cells primarily by trogocytosis and cross-dressing, whereas CD169+ macrophages took up antigen mostly from apoptotic cells by phagocytosis; and that phagocytosis of apoptotic cells was the primary priming mechanism for apoptotic/irradiated cells. The 'don't-eat-me' molecule CD47, which engages the inhibitory receptor SIRPα on phagocytes and is commonly overexpressed by tumor cells, is the one signal known to bias phagocytes against engulfing live cells, but the broader molecular logic of the trogocytosis-versus-phagocytosis decision remained unidentified.

These findings sharpen a mechanistic puzzle. Phagocytosis of apoptotic corpses (efferocytosis) is well documented to deliver tolerogenic, immunosuppressive signals to DCs, which would explain why live-cell antigen is superior for cross-priming — but this explanation was inferential. Moreover, measuring how much cross-dressing contributes in vivo is technically hard because DCs express the same MHC alleles as every other host cell, so peptide–MHC generated inside the DC cannot be distinguished from peptide–MHC acquired from a tumor cell without bone-marrow chimeras (mice whose immune cells derive from transplanted donor marrow carrying distinguishable MHC alleles) or conditional MHC class I knockout alleles (Cre-regulated alleles deleting MHC-I only in defined cell types), tools that only became available recently.

Problem Statement

The problem arising from the findings of Das Mohapatra et al. (2020) and taken up by the subsequent literature is to determine mechanistically how CD8α+ dendritic cells acquire antigen from live tumor cells and how much this route contributes to cross-priming in vivo. Its linked components are: (1) identify the precise molecular signals on live tumor cells and on the DC that direct the DC to trogocytose membrane fragments (leading to cross-dressing with preformed peptide-MHC and, separately, to transfer of membrane-associated antigens) rather than to phagocytose the cell — beyond the known overexpression of the CD47 'don't-eat-me' signal; (2) establish the mechanistic basis for the superior immunogenicity of live-cell antigen, in particular whether trogocytosis and cross-dressing intrinsically avoid the tolerogenic signaling that phagocytosis of apoptotic corpses induces in DCs, or whether other factors account for the difference; (3) quantify the relative contribution of cross-dressing versus other antigen-acquisition and cross-presentation pathways — uptake of heat shock protein–peptide complexes (gp96/CD91), transfer of intact protein antigens, phagocytosis-dependent classical cross-presentation — across different tumor models and anatomical locations in vivo; and (4) from that mechanistic understanding, evaluate the feasibility and optimal design of dendritic cell-based immunotherapies that use live autologous tumor cells as the source of cross-dressable antigen, in preclinical and clinical settings. These components are linked open questions serving one objective — a causal, quantitative account of live-cell cross-priming: components (1), (2), and (3) constitute that account, and component (4) is a therapeutic application of it rather than a separate required part.

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