As a result of ongoing research, the system by which DCs are classified continues to change. Currently, as new subsets are discovered, characterization often places them into one of two groups: steady state conventional DCs (cDCs) or non-conventional DCs (Kushwah and Hu, 2011). This initial classification is based on lineage, function, and location, with each subset being identified by the presence or absence of different cell surface markers. Steady state cDCs, as the name suggests, are present and function continually, even during inflammation, and include subsets found in the lymphoid organs as well as migratory subsets present in the tissues. Non-conventional DCs are mostly comprised of monocyte-derived DCs subsets, populations which are highly enriched during inflammation and thus are often referred to as inflammatory DCs (Shortman and Liu, 2002; Heath et al., 2004; Shortman and Naik, 2007; Liu et al., 2009; Kushwah and Hu, 2011). DCs found in non-lymphoid tissue regardless of whether formally categorized as steady state conventional or monocyte-derived non-conventional, are typically classified by the tissue in which they are found and the presence or absence of CD103, CD11b, langerin (for skin associated subsets), or the chemokine receptor CX3CR1 (Kushwah and Hu, 2011). Though some of these subsets are capable of cross-presentation, this review focuses on those subsets present in the spleen. We refer those interested to several publications that more completely dissect the lineages, functionality and surface expression of various markers in these other DC populations (Shortman and Liu, 2002; Heath et al., 2004; Shortman and Naik, 2007; Liu et al., 2009; del Rio et al., 2010; Liu and Nussenzweig, 2010; Shortman and Heath, 2010; Kushwah and Hu, 2011). Plasmacytoid DCs (pDCs), a subset also present in the spleen during steady state, are functionally distinct from both cDCs and non-conventional subsets, but possess a common precursor with cDCs subsets. In spite of this connected lineage, functional differences between cDCs and pDCs complicates the exact placement of the latter subset and, thus, has led to controversy. As a result, some researchers place pDCs with non-conventional DCs while others place them within a distinct group called pre-DCs or within their own category (Shortman and Liu, 2002; Shortman and Naik, 2007; Liu and Nussenzweig, 2010; Kushwah and Hu, 2011).

Conventional DCs and pDCs present in spleen and lymph nodes are distinguished by differential expression of CD11c, B220, and PDCA-1. Splenic cDCs lack B220 and PDCA-1 (Figure 1A) and can be further divided into four subpopulations characterized by the presence or lack of various markers (Table 1): (1) CD8α DCs (CD8α⁺, CD4⁻, CD11b⁻); (2) CD11b DCs (CD8⁻, CD4⁻, CD11b⁺); (3) CD4 DCs (CD8⁻, CD4⁺, CD11b⁺); or (4) merocytic DCs (mcDCs)/CD8⁻, CD4⁻, CD11b⁻ DC (Figure 1A; Janssen et al., 2006; Reboulet et al., 2010; Shortman and Heath, 2010; Hennies et al., 2011; Kushwah and Hu, 2011). We have investigated, and thus will discuss in this review, the uptake of cell-associated Ag under steady state conditions and their cross-presentation within four splenic DC subgroups – CD8α DCs, CD11b DCs (which includes the CD4⁺ subset), mcDC/CD8⁻, CD4⁻, CD11b⁻ DC, and pDCs.

The CD8α DC, classically considered to be the major cross-presenting DC subset in the mouse spleen, is located in the T cell zone of the spleen and has repeatedly been shown to effectively cross-present beads, soluble Ag, and cell-associated Ag (Figures 1B,C; den Haan et al., 2000; Pooley et al., 2001; Heath et al., 2004; Belz et al., 2005; Schnorrer et al., 2006). The development of CD8α DCs is dependent on Flt3L-STAT3 signaling. While cDC development in general requires the transcription factors Ikaros, and PU.1 (Wu et al., 1997; Anderson et al., 2000; Guerriero et al., 2000; Allman et al., 2006; Wu and Liu, 2007), the CD8α DC lineage commitment is also dependent on Batf3, IRF-8, and Id2 (Schiavoni et al., 2002; Hacker et al., 2003; Hildner et al., 2008). This DC subset, in addition to being CD8α⁺, also expresses DEC205, XCR1, and Clec9a (Vremec et al., 2000; Sancho et al., 2009; Shortman and Heath, 2010; Crozat et al., 2011). Depending on age and strain of mouse, up to 70% of CD8α DCs co-express CD103, which has been suggested to represent a developmental stage or activation state within the CD8α DC population or a CD8⁺ subset with distinct functionality (Pribila et al., 2004; Qiu et al., 2009; del Rio et al., 2010; Shortman and Heath, 2010). CD8α DCs take up dead cells more readily than other splenic DC subsets (Figure 1A; Iyoda et al., 2002; Schulz and Reis e Sousa, 2002; Schnorrer et al., 2006) and have been implicated in the induction and maintenance of CD8⁺ T cell tolerance to cell-associated Ag in models of autoimmunity and transplantation (Kurts et al., 1996, 1998; Hawiger et al., 2001; Belz et al., 2002; Bonifaz et al., 2002; Scheinecker et al., 2002; Shortman and Heath, 2010). This subset’s importance in the cross-presentation of cell-associated Ag is further supported by a dramatic reduction of anti-tumor immunity in mice deficient in Batf3, a gene crucial for the development of the CD8α DC precursor (Hildner et al., 2008).

Splenic CD11b DCs reside in the marginal zone of the spleen and predominantly co-express CD4, DCIR2, and Sirp-α (Crowley et al., 1989; Pulendran et al., 1997; Maldonado-Lopez and Moser, 2001; Lahoud et al., 2006). CD11b DC development is governed by transcription factors IRF2/4 (Honda et al., 2004; Ichikawa et al., 2004; Suzuki et al., 2004) and RelB (Burkly et al., 1995; Weih et al., 1995; Wu et al., 1998).

While CD11b DCs display great potential for phagocytosis of proteins, beads/particles, and bacteria, their capacity for cross-presentation under steady state conditions is poor. Moreover, CD11b DCs display weak phagocytosis of apoptotic cells and no role has been described for these cells in cross-presentation to cell-associated Ag under steady state conditions (Figures 1A–C; den Haan et al., 2000; Pooley et al., 2001; Iyoda et al., 2002; Schulz and Reis e Sousa, 2002; Morelli et al., 2003; Schnorrer et al., 2006).

Splenic pDCs are defined by strong expression of both B220 and PDCA-1 and are predominantly located in the T cell area and red pulp. While there is some discussion on the exact delineation of pDC with regard to shared precursors with other cDC, research has shown the requirement for the transcription factors E2-2, IRF8, and Spi-B (Schiavoni et al., 2002; Schotte et al., 2004; Cisse et al., 2008). Although pDCs are poor at taking up cell-associated Ag, depletion studies have shown that pDCs are critical in the induction of tolerance after intravenous injection of apoptotic cells. Such tolerance, however, does not require direct pDC–apoptotic cell interactions, but rather soluble mediators from marginal zone macrophages (Bonnefoy et al., 2011).

Over the recent years various laboratories have identified splenic DCs that lack the conventional markers (CD8α⁻, CD11b⁻, CD4⁻; Figures 1A–C; Hochrein et al., 2001; Naik et al., 2005; Janssen et al., 2006; Vremec et al., 2007; Bedoui et al., 2009a; Katz et al., 2010; Reboulet et al., 2010; Hennies et al., 2011). Generally, these populations are relatively small and only comprise <1–10% of the DC in a naïve steady state spleen. Several of these DC populations have been shown to cross-present antigens in protein (Vremec et al., 2007) or cell-associated form (Bedoui et al., 2009a; Reboulet et al., 2010; Hennies et al., 2011). Flt3L treatment of mice significantly increases the frequency of these DCs, and cells with similar features can be generated by in vitro Flt3L bone-marrow cultures (Bedoui et al., 2009a; Reboulet et al., 2010; Hennies et al., 2011).

During the course of our work we have named the CD8α⁻ CD11b⁻ CD4⁻ DC in the naïve spleen mcDCs due to the smaller particles (meros = particle) characteristically taken up by these cells (Figure 2C) and will use this name throughout this review (Reboulet et al., 2010). Like CD8α DCs, mcDCs express XCR1, Clec9a, and are Sirp-α negative, but in contrast are DEC205⁻, CD103⁻, and CD11b^−/dull (Table 1). It has been suggested that this marker negative subset is a precursor to the CD8α DCs (Janssen et al., 2006; Bedoui et al., 2009a), a hypothesis supported by the presence of Clec9a and CD24, surface molecules shown to be present on the immediate precursors of CD8α DCs that lack CD8 and DEC205 expression (Sathe and Shortman, 2008; Shortman and Heath, 2010; Kushwah and Hu, 2011). CD8α⁻ CD11b⁻ CD4⁻ DC obtained from Flt3L treated mice readily convert into CD8α DCs upon transfer (Bedoui et al., 2009a). However, only a small fraction of CD8α⁻ CD11b⁻ CD4⁻ DC/mcDC from naïve spleens convert to CD8α DCs (Reboulet et al., 2010) suggesting that mcDCs are either “long-term” CD8α DC precursors that are relatively resistant to conversion or a stable population that possesses unique functionality and marker expression compared to other known precursors.

Merocytic DCs take up cellular material from dead and dying cells, though be it less than CD8α DCs (Figure 1A). Though mcDCs take up less apoptotic cell material they show extended cross-priming of CD8⁺ T cells due to prolonged storage of cell-associated Ag (Reboulet et al., 2010). Importantly, mcDCs prime both CD4⁺ and CD8⁺ T cells to cell-associated Ag (Figures 1B,C; Janssen et al., 2006; Reboulet et al., 2010). CD4⁺ T cell activation is important in the induction of immunity against cell-associated Ag as CD8⁺ T cells become tolerant without sufficient CD4⁺ T cell help (Janssen et al., 2003, 2005; Griffith et al., 2007). CD8⁺ T cells primed by mcDC to cell-associated Ag show greater capacity for primary expansion, cytokine production, and memory formation on a per cell basis than those primed by CD8α DC (Janssen et al., 2006; Katz et al., 2010; Reboulet et al., 2010; Hennies et al., 2011).

Merocytic DC have been associated with the breaking of tolerance and acceleration of immune responses to cell-associated Ags. Treatment of tumor bearing mice with mcDC previously exposed to irradiated tumor cells, resulted in tumor suppression and increased host survival through the activation of naïve tumor-specific CD8⁺T cells as well as the reinvigoration of tumor-specific T cells that had been rendered non-responsive by the tumor in vivo (Reboulet et al., 2010). Dysregulation of the mcDC compartment has also been associated with the development of autoimmunity; mcDCs are more numerous and more biologically active in the non-obese diabetic (NOD) mouse model of type I diabetes and absolute numbers correlate with disease development and progression. Transfer of mcDCs – loaded with irradiated islet cells– transferred diabetes in young NOD recipients. Moreover, when purified from the pancreatic lymph nodes of overtly diabetic NOD mice, mcDCs break peripheral tolerance to beta antigens in vivo and induce the rapid onset of T cell-mediated type I diabetes in young NOD mice (Katz et al., 2010).

As a cell becomes apoptotic, the steady state “don’t eat me” signals of viable cells are lost and replaced through a series of morphological and biochemical changes (Elward and Gasque, 2003; Erwig and Henson, 2008; Poon et al., 2010). The most prominent and perhaps best-characterized change is the exposure of phosphatidylserine (PS) on the surface of the dying cell. Once PS is exposed, it can be recognized by a number of bridging molecules including milk fat globular-EGF factor 8 protein (MFG-E8; Borisenko et al., 2004; Hanayama et al., 2004), growth arrest-specific 6 (Gas6; Ishimoto et al., 2000; Scott et al., 2001), β2-glycoprotein I (β2-GPI; Balasubramanian et al., 1997), and serum Protein S (Erwig and Henson, 2008; Krysko and Vandenabeele, 2008; Poon et al., 2010). Many of these bridging molecules then facilitate recognition by receptors on the surface of the phagocyte, including the integrins αvβ3 or αvβ5 (Borisenko et al., 2004; Hanayama et al., 2004; Erwig and Henson, 2008). Additionally, the apoptotic cell and other bridging molecules like TSP-1 are recognized by phagocytic receptors such as Tim-1, Tim-3, and Tim-4 (Kobayashi et al., 2007; Nakayama et al., 2009; Albacker et al., 2010), CD36 (Albert et al., 1998), Treml2 and Treml4 (Hemmi et al., 2009), DEC205 (Shrimpton et al., 2009), class A scavenger receptors (Platt et al., 2000), Lox-1 (Oka et al., 1998), and various C-type lectins including Clec9a (DNGR1; Krysko and Vandenabeele, 2008; Sancho et al., 2009; Poon et al., 2010; Shortman and Heath, 2010). While the seemingly overabundance of receptors involved in dying cell uptake stresses the importance of apoptotic cell removal in the maintenance of immune homeostasis, it is becoming apparent that these molecules are not merely redundant, but may distinctly influence APC behavior, and, thus, guide specific responses toward cell-associated antigens under various settings (Bratton and Henson, 2008; Erwig and Henson, 2008; Krysko and Vandenabeele, 2008).

Cross-presentation, but not necessarily cross-priming, of cell-associated Ag is generally enhanced when uptake is mediated by DEC205 (Bonifaz et al., 2002), Clec9a (Sancho et al., 2008, 2009), Lox-1(Delneste et al., 2002), or Fc gamma R (Regnault et al., 1999; Rodriguez et al., 1999; Amigorena, 2002; den Haan and Bevan, 2002; Kalergis and Ravetch, 2002; Flinsenberg et al., 2011). Uptake via the mannose receptor has also been implicated in cross-presentation pathways, but its relevance has thus far only been shown within the context of soluble Ag, not cell-associated Ag (Burgdorf et al., 2006, 2008). Comparison of the transcriptome of the four DC subsets suggests the most similarity between mcDCs and CD8α DCs with regard to the expression of molecules involved in the recognition and uptake of dying cells. However, some receptors are still restricted to one subset – as is the case for DEC205 and CD8α DCs – or have differential expression/different levels of expression, resulting in unique patterns for each subset (Table 1).

Consistent with receptor-mediated uptake leading to cross-presentation, both CD8α DCs and mcDCs appear to take up cellular material via a classical receptor-mediated phagocytic process. Transmission electron microscopy (TEM)of CD8α DCs and mcDCs exposed to dying cells predominantly shows the presence of small particles of phagocytosed material tightly surrounded by a double membrane (Figure 2A). In contrast, CD11b DCs appear to use a more macropinocytic mechanism resulting in the uptake of larger particles and inclusion of extracellular solutes and fluids (Figures 2A,C).

Our studies and the work of others indicate that the method of uptake, i.e., receptor-mediated phagocytosis vs. macropinocytosis, influences the particle size taken up by APCs (Rejman et al., 2004). While particle size determines the total amount of Ag available to the cell, a growing body of literature indicates that particle size also affects intracellular trafficking, the kinetics of phagosomal pH, and thereby cross-presentation (Fifis et al., 2004; Rejman et al., 2004; Tran and Shen, 2009). Cross-presentation has been shown to be enhanced when Ag are bound onto particles between the range of 0.5 and 3 μ (Tran and Shen, 2009). However, it is likely that the optimal size for cross-presentation will be affected by the composition of the particle, the receptors involved in the uptake and the nature of the cell.

Transmission electron microscopy combined with ImageStream technology, a flow cytometric based method that allows for quantitative image analysis on vast number of cells, confirmed that cross-presenting splenic DCs differentially take up material from dead and dying cells as measured by total particles per cell and the overall particle size. CD8α DCs and mcDCs not only take up particles of dying cells more readily, but also preferentially take up smaller particles than CD11b DCs (Figures 2B,C), a size differential that most likely facilitates the entrance of cell-associated Ag into cross-presentation pathways (Fifis et al., 2004; Rejman et al., 2004; Tran and Shen, 2009).

Interestingly, upon exposure to dead and dying cells, mcDCs generally take up a comparable number of particles/cell to CD8α DCs, but these particles are typically smaller in size (Figures 2B,C; Reboulet et al., 2010). While the mcDCs takes up a lower “net amount” of Ag, the smaller particle size might expedite the export to the cytosol which would facilitate Ag processing (Rodriguez et al., 1999; Rock et al., 2010).

Upon uptake, cellular material from dying cells is found within early phagosomes – or sorting endosomes – characterized by the presence of the early endosomal markers EEA-1, Rab5, PI(3)P, syntaxin 13, transferrin, and vesicle-associated membrane protein 3(VAMP-3; Vieira et al., 2002; Peng and Elkon, 2011). The phagosome is transformed into a phagolysosome through a progressive maturation process that is dependent on the sequential fusion of endosomes and lysosomes with the internalized phagosome. Most recently this maturation process and antigen cross-presentation was shown to be regulated by the SNARE protein Sec22b through its control of ER-resident protein recruitment to phagosomes (Cebrian et al., 2011). Late endosomes/late phagosomes are associated with Rab7, Rab9, mannose 6-phosphate receptor, syntaxin7, LAMP-1 and LAMP-2 (Vieira et al., 2002). The final product, phagolysosomes, express LAMPs but have lost most of the earlier endosomal markers. In addition, the phagolysosomes possess a number of complementary degradative properties, including a very low pH, hydrolytic enzymes for particle digestion, defensins and other bactericidal peptides, and the ability to generate toxic oxidative compounds (Amigorena and Savina, 2010; Rock et al., 2010).

Cellular material taken up by CD11b DCs rapidly ends up in fully matured phagolysosomes (Figures 3A–C; Savina et al., 2006, 2009; Reboulet et al., 2010; Peng and Elkon, 2011). Colocalization studies show an association of phagocytosed materials with LAMP-1+ organelles and pulse-chase experiments show the degradation of >80% of the material in less than 20 h (Figures 3A,B). As a result, Ag are quickly processed and either shuttled into MHC class II restricted pathways or simply degraded and disposed of. In contrast, cross-presenting DC subsets take up material from dying cells and hold these particles in immature endosomes for an extended period of time (Figures 3A–C). While CD8α DCs have degraded most of the material after 20 h, digestion is much slower than in CD11b DCs. Phagocytosed cellular particles in mcDCs are more colocalized with EEA-1 and less with LAMP-1 after 4 h of co-incubation with irradiated cells, indicating slower endosomal maturation (Figure 3A). Moreover, pulse-chase studies showed the persistence of materials over a 20-h time span (Figure 3B; Reboulet et al., 2010). This persistence of Ag in the mcDCs has been shown to prolong the cross-presentation of specific cell-associated Ag and thereby increase T cell priming (Savina et al., 2006; Reboulet et al., 2010; Peng and Elkon, 2011).

As endosomal acidification causes the robust activation of lysosomal proteases and the subsequent destruction of Ag, acidification is considered to be poorly compatible with cross-presentation. Forced lysosomal acidification dramatically reduces cross-presentation while prevention of acidification has been shown to enhance cross-presentation (Savina et al., 2006; Amigorena and Savina, 2010; Reboulet et al., 2010). Though it was previously described that DCs have relative ineffective acidification of their lysosomes, the mechanism under pinning the sustained phagosome alkalinization was only recently unraveled. Studies by laboratory of Mellman and Amigorena indicate that this results from an incomplete assembly of V-ATPase in DC lysosomes and the Rab27-mediated recruitment of the NADPH NOX2. The NOX2-mediated generation of reactive oxygen species (ROS) in endocytic compartments causes the consumption of protons, followed by the active alkalinization of these compartments (Trombetta et al., 2003; Savina and Amigorena, 2007; Savina et al., 2009; Rock et al., 2010).

Acidification studies indicate that endosomes with cell-associated material in both CD8α DCs and mcDCs maintain a similar high/neutral pH for several hours post uptake of cell-associated antigens, which correlates with Ag persistence. Compared to CD8α DCs, mcDCs show decreased lysosomal acidification over a prolonged period of time, resulting in a less acidic endosomal compartment after 20 h (Figures 3B,C; Reboulet et al., 2010).

In both populations the treatment with diphenylene iodonium (DPI) – an inhibitor of flavin-containing enzymes such as NOX2 – accelerates lysosomal acidification, prevents Ag persistence, and rapidly decreases the cross-presenting capacity of both CD8α DCs and mcDCs, emphasizing the importance of endosomal acidification in their cross-presentation (Reboulet et al., 2010).

The mechanisms that govern the prolonged Ag persistence in mcDC remain unclear as the biogenesis of phagolysosomes still involves many poorly understood processes. Transcriptome analysis of CD8α DCs and mcDCs showed ≈20-fold higher expression of Cybb (NOX2) in mcDCs. In addition, differential expression of various R- and Q-SNAREs (soluble N-ethyl maleimide sensitive-factor attachment protein receptors), sorting nexins, and V-ATPases that have been suggested to play a role in vesicle transport and fusion are seen (Vieira et al., 2002; Cebrian et al., 2011). However, as most of these processes depend on active recruitment of these proteins to endosomal/lysosomal membranes, differences in expression levels might not be indicative of their degree of involvement. It is more likely that the nature of the phagocytosed particle – including size – and the receptors involved in their uptake dictate phagosome maturation (Peng and Elkon, 2011).

As intact internalized Ag fill the cell, there are two proposed pathways by which they are cross-presented: the vacuolar and cytosolic pathways. The vacuolar pathway hypothesizes that cross-presented Ag are fully processed within the endosomes. The aminopeptidase IRAP facilitates the production of MHC class I-specific peptides that bind to the MHC molecule within the endosome. This pathway appears to be cathepsin S dependent and TAP independent (Shen et al., 2004; Chen and Jondal, 2008; Rock et al., 2010). In contrast, the cytosolic pathway requires minimally processed antigen to escape into the cytosol. Once in the cytosol, the Ag is processed by the proteasome, and generated peptides are shuttled into the lumen of the ER via sec61 or into phagosomes that have recruited ER components. The ER associated aminopeptidase ERAP actively clips the peptides to the proper length and TAP facilitates the loading into MHC class I (Rock et al., 2010).

Multiple studies indicate a dominant role for the cytosolic pathway in the processing of cell-associated Ag by cross-presenting DC subsets (Figure 4; Shen et al., 2004; Rock et al., 2010). Smaller particles, like those taken up by mcDCs and CD8α DCs (Figure 2C), are more rapidly and efficiently exported to the cytosol, a process that would drive the cytosolic pathway of cross-presentation (Rodriguez et al., 1999). Also, lactacystin and brefeldin A, inhibitors of the proteasome and Golgi transport, respectively, completely inhibit the ability of both CD8α DCs and mcDCs to activate Ag-specific CD8⁺ T cells against cell-associated Ag (Figure 4). A recent report by Cebrian et al. (2011) implicates the SNARE protein Sec22b as an essential element of the cytosolic pathway. Depletion of Sec22b inhibits the recruitment of ER-resident proteins to the phagosome and phagolysosomal fusion was enhanced. As a result, antigens are rapidly degraded instead of being transported to the cytosol. These combined effects of Sec22b loss drastically reduce the cross-presentation of soluble and surface bound OVA as well as parasite and bacterial associated antigens. Interestingly, the presentation of both MHC class II and endogenous MHC class I restricted peptides is unaffected demonstrating significant separation between these processing pathways (Cebrian et al., 2011). Taken together, these data support the hypothesis that Ag must reach the cytosol, and eventually become associated with ER-derived proteins, to be cross-presented.

The maintenance of a more neutral pH correlates with delayed or reduced lysosomal protease activation. Cross-presenting DCs characteristically express lower levels of proteases and higher levels of protease inhibitors as compared to other APCs (Lennon-Dumenil et al., 2002; Trombetta et al., 2003). Pepstatin A, an inhibitor of acid proteases involved in lysosomal maturation and acidification, has no effect on the DCs capacity to cross-present. This demonstrates that protease mediated peptide production plays a minimal role in cross-presentation (Figure 4; Rock et al., 2010). Interestingly, leupeptin, an inhibitor of cysteine proteases essential for the vacuolar pathway, partially inhibits cross-presentation, but affected the mcDCs more than the CD8α DC subset. This suggests that mcDCs may utilize the vacuolar pathway for the processing of cell-associated Ag more than other subsets. In support of this, removal of either ERAP or IRAP results in only a 50% reduction in cross-presentation, suggesting the utilization of both pathways by cross-presenting DCs (Firat et al., 2007; Blanchard et al., 2008; Saveanu et al., 2009; Rock et al., 2010).