Conventional dendritic cells (cDCs) are the most common DC subset (43, 69). cDCs can be identified based on their location: lymphoid resident cDCs sample antigens through draining lymph, while peripheral cDCs traffic to lymph nodes after capturing antigen. Within lymphoid tissues, such as lymph nodes, they can then present antigen to and activate naïve T cells. As with all DCs, cDCs commonly express CD11c and MHC II, but these markers also arise in pDCs, and on macrophages at lower levels (43).

CD24 and CD26 are potential pan-cDC surface targets, but are also present on a wide variety of immune and stromal cell types (31, 70–75). Antibody targeting of these receptors have been used in contexts outside vaccination, and could potentially enable a platform to target cDCs (75–77). However, given their expression in other tissues, they may not provide selective targeting of cDCs.

Other cDC markers are used to delineate the two subsets of cDCs, conventional type 1 and type 2 DCs (cDC1s and cDC2s). There are two important clarifications for cDC subtypes. First, while expression of transcription factors and surface molecules is a widely accepted method of classification, recent research has shown heterogeneity in these populations, especially during active infection (78). Furthermore, recent single cell sequencing studies suggest other categories of cDCs, particularly in humans, with nuanced lineages and functions (79, 80). As we are focusing on receptor targeting and consequences on downstream effector responses, rather than the intricacies of innate immune classification, here we will rely on established cDC1 and cDC2 markers and briefly overlook newer paradigms of classification for simplicity.

With regard to vaccine design; cDC1s and cDC2s function differently when activating T cells, making them potential targets for vaccines which can modulate T cell responses (43). In mice, cDC1s are generally associated with type 1 immune responses, including cross-presentation of exogenous or cell-associated antigen on major histocompatibility complex class I (MHC I). As a result, cDC1s primarily facilitate CD8⁺ T cell responses and release Th1-biasing cytokines (33, 41, 81, 82). cDC2s are generally associated with type 2 and type 3 immune responses, including capturing soluble antigen. As a result, cDC2s primarily facilitate CD4⁺ T cell and antibody responses (33, 41, 81–84). However these delineations are less strict in human cDCs, and under certain conditions human cDC1s can facilitate type 2 immune responses, while conversely human cDC2s can facilitate CD8⁺ T cell and type 1 immune responses (41, 85, 86). The next sections will discuss cDC1 and cDC2 targeting in detail.

pDCs are a small subset of DCs that circulate through the blood and are specialized for viral recognition and response. They express TLRs which recognize foreign nucleic acid, such as TLR7 and TLR9, and rapidly secrete type I and type III interferons (IFNs) in response to infection (31, 40, 156, 157). pDCs are typically identified by expression of B220 (CD45R), Ly6C, Siglec-H, and BST2 (CD317) in mice, BDCA2 (CD303), BDCA4 (CD304), and CD123 (IL-3R) in humans, and CD45RA in both (31, 40). Receptors that have been used for pDC targeting include PDC-TREM, Siglec-H, BST2, DEC-205, DCIR, BDCA2, and FcγRII (55, 158–164).

The main pDC receptors studied for targeted vaccination are PDC-TREM, Siglec-H, and BST2. PDC-TREM is a pDC-specific receptor that triggers type I IFN production (165). Siglec-H is a pDC-specific receptor that functions as an endocytic receptor, but unlike other receptors in the Siglec family, it does not bind sialic acids (162). Pugholm et al. showed that immunization with rat anti-PDC-TREM without adjuvant was able to raise anti-rat IgG, while anti-Siglec-H was unable to raise an antibody response (55). Targeting of Siglec-H by mAb-antigen conjugates without adjuvant led to a tolerogenic CD4⁺ T cell response (163), but in the presence of adjuvant a CD8⁺ T cell response was generated (162). BST2 is a receptor that prevents virion dissemination by binding virion lipid membranes (166). BST2 identifies pDCs under homeostatic conditions, but is upregulated on most cells during viral infection and inflammation (167). Similarly, targeting of BST2 by mAb-antigen conjugates in the presence of adjuvant generated a CD8⁺ T cell response and IgG2 production, suggesting a Th1 response (164). However, CD8⁺ T cell responses from pDC targeting is thought to occur through antigen transfer to cDC1s and not directly from pDC-T cell interactions (168). Taken together, this suggests that while PDC-TREM targeting can raise antibody responses without adjuvant, Siglec-H and BST2 targeting require adjuvant and potentially other DC subsets to generate humoral and T cell responses.

Due to their availability in blood, pDCs are more extensively studied in humans than other DC subsets. Human pDCs have been targeted via DEC-205, DCIR, BDCA-2, and FcγRII (158–161). Tel et al. demonstrated that when PLGA nanoparticles containing OVA and R848 were targeted to pDCs via these receptors, CD8⁺ and CD4⁺ T cell responses were generated (158). Human pDC-generated immune responses are likely dependent on the presence of adjuvant, as targeting and ligation of DCIR and BDCA2 alone can suppress DC activation (159, 160). To this end, Sepulveda-Toepfer et al. generated an αFcγRII-CpG conjugate that was shown to target and activate human pDCs (161); however, its efficacy in a vaccine formulation has yet to be assessed. Additionally, the presence of FcγRII on other immune cells such as cDC1s and its previous use as a cDC1 target, as well as the activating and inhibitory isoforms of this receptor, should be considered (101, 119–122). Overall, the literature indicates that targeting pDCs can generate type 1 immune responses, but the response can vary depending on the targeted receptor and on the presence of adjuvant. More research is required to fully establish the effects of pDC targeting during vaccination.

Formulations administered via subcutaneous and intramuscular injection routes will encounter other types of cDCs that should be considered in the design of effective vaccines. Under steady state conditions, cDCs can be further classified into tissue resident and migratory subsets. This classification is determined on if they primarily reside in local tissue environments or are primed to sample antigens from local tissues and migrate to nearby lymphoid tissues (69). DC phenotypes can further vary by tissue location, such as lung and intestinal DCs. The classification and functions of these tissue-resident DCs have been reviewed extensively elsewhere (31, 73, 87, 142, 169–172); this review is focused on APCs in lymphoid tissues. In lymphoid tissues, migratory cDCs express more DC maturation markers, and have higher levels of MHC II and lower levels of CD11c compared to resident cDCs under homeostatic conditions (31). However, under inflammatory conditions, where both resident and migratory DCs will mature, this classification no longer holds (31). Instead, lymphoid resident and migratory cDC1s can be identified by expression of CD8α or CD103, respectively (31, 87). Semmrich et al. showed that targeting CD103⁺ DCs via mAb-antigen conjugates raised both CD8⁺ and CD4⁺ T cell responses in the presence of adjuvant (173). In the absence of adjuvant, a tolerogenic response resulted (173). CD103 is an integrin that plays a role in tissue residence for a wide variety of cell types, including T cells (174), and thus is a non-specific target for migratory cDC1s. However, the immunogenic response is consistent with the importance of CD103⁺ DCs in generating CD8⁺ T cell responses (175). The contrasting immunogenic and tolerogenic responses are further consistent with the targeting of cDC1s via receptors such as DEC-205, where the presence of adjuvant determines the immunogenic or tolerogenic nature of the response (93–95, 97). No markers have yet been identified to distinguish lymphoid resident and migratory cDC2s. Further delineation of resident and migratory DCs and their functions have been reviewed in depth (31, 87, 169); however in the context of targeted vaccines, DCs present at the site of vaccination that are primed to migrate prior to antigen encounter should be considered and further studied.

One unique tissue-resident class of DCs are the Langerhans cells (LCs). LCs are specialized, skin-resident, immature APCs that reside in the skin and can be identified by the presence of cytoplasmic organelles, known as Birbeck granules (176–181). They are also distinguished by the expression of CD207 (Langerin) (176–181). While Langerhans cells share common features of DCs, including the characteristic dendrite projections and expression of CD11c and MHC II, they arise from macrophage progenitors and monocytes (176–178, 180, 181). Thus, while LCs have been classically considered to be a DC subset, some have proposed considering LCs as a macrophage subset (179, 181). Furthermore, studies have observed CD207-expressing DCs that are distinct from LCs, making CD207 a less reliable LC target (177, 178, 180). Regardless, as LCs exist in the skin and encounter vaccine antigens delivered subcutaneously, some studies have investigated targeting vaccines to LCs or CD207⁺ DCs (182). Li et al. targeted LCs using mannose ligands, which bind CD207, conjugated to melanoma antigens. In this study, poor induction of a CD8⁺ T cell response compared to untargeted antigen was observed (183). Bellmann et al. conjugated viral proteins to anti-CD207 or anti-DEC-205 mAbs and directly compared their efficacy in promoting T cell responses in human ex vivo skin models (184). Similarly, the CD207-targeted vaccines were ineffective in generating CD8⁺ or CD4⁺ T cell responses, while the DEC-205-targeted formulation generated a moderate T cell response as reported by others (184). These results are consistent with previous findings that dermal DCs, and not LCs, are the primary drivers of immune responses in skin (185). Despite these findings, Carter et al. hypothesize that Th1-biased immune responses are driven by Langerhans cells in the skin (131). They show that CD8⁺ T cell responses are enhanced when targeting cDC2s via Dectin-1, which is also expressed by LCs, after subcutaneous injection (131, 146). Given the clear challenges in classifying migratory and resident DCs in the skin, further investigation is required to understand the relative contributions of dermal DCs and Langerhans cells in vaccine-mediated immunity after subcutaneous injection.

Macrophages are often classified into “pro-inflammatory” M1 and “anti-inflammatory” M2 phenotypes; M1 macrophages express high levels of MHC II, CD40, CD80, CD86, and TLR4, while M2 macrophages express CD163, CD206, MARCO (CD204), Dectin-1 (Clec7a), and DC-SIGN (CD209) (199, 207, 208). However, these phenotypes are no longer considered discrete states but rather a spectrum, as macrophages may switch from one phenotype to another or simultaneously express markers that define M1 and M2 states (35, 197, 199–201, 203, 204, 207, 208). M1 macrophage targeting has generally focused on polarization towards M2, particularly for applications in treating inflammatory diseases (209). Conversely, M2 targeting has focused on polarization towards M1 or specific M2 depletion. Such targeted responses are of value for cancer therapy, as tumor-associated macrophages (TAMs) are phenotypically similar to M2 macrophages (208, 210), and increased levels of M2 TAMs are associated with poor outcomes (203). However, since “repolarization” strategies often focus on bulk macrophage populations and given the plasticity of the M1/M2 spectrum, specific M1/M2 targeting has, to date, been rare.

We highlight here some examples of macrophage targeting, which have primarily been used for macrophage reprogramming. Jain et al. demonstrated reprogramming of M1 macrophages to M2 using IL-10 encoding DNA loaded into alginate nanoparticles bearing tuftsin peptide, which broadly enhances phagocytosis by macrophages (211, 212). Farajzadeh et al. and Tran et al. demonstrated reprogramming by delivering M2-polarizing therapeutics in hyaluronic acid-based nanoparticles, which bind CD44, a marker overexpressed on macrophages (213, 214). However, both these strategies rely on pan-macrophage marker targeting strategies, rather than targets that achieve M1 or M2-specific delivery.

Alvarado-Vasquez et al. reprogrammed M1 macrophages to M2 by delivering CD163 encoding plasmids via a mannose-grafted polyethyleneimine (PEI) nanoparticle, targeting CD206 (215). Conversely, Zhu et al. targeted M2 TAMs using a mannose-grafted PLGA nanoparticle (216), demonstrating the difficulty of M1/M2 target specificity. Similarly, although Dectin-1 is considered a M2 marker, β-1,3-D-glucan nanoparticles targeting Dectin-1 have been used to deliver TNF-α-suppressing siRNAs to pro-inflammatory macrophages (217, 218). However, Cieslewicz et al. demonstrated specific targeting of M2 TAMs, generating a M2-specific targeting peptide by phage display against M2 macrophages to deliver pro-apoptotic peptides (210). This targeting ability and specific depletion could be improved by multivalency (219). Lee et al. also demonstrated M2-specific targeting and depletion using melittin, an amphiphilic peptide derived from honey bee venom, after conjugation to pro-apoptotic peptides (220, 221). M2 TAMs are also known to overexpress folate receptor beta (FRβ), and its natural ligand, folic acid, may be used for M2 TAM targeting (222). Hattori et al. and Tie et al. demonstrated that folate-decorated liposomes were able to target M2 TAMs, while Nagai et al. demonstrated M2 TAM targeting using an FRβ ScFv (223–225). Due to the focus of the field being on altering M1/M2 phenotype switching or M2 depletion, rather than targeting native M1 or M2 macrophages, the effect of M1/M2 targeting on adaptive immune responses remaining an open question and an opportunity for future research.

Another macrophage subset with implications for targeted vaccines is CD169⁺ macrophages. CD169 (Siglec-1) is a sialic acid binding receptor expressed on macrophages from lymphoid tissues (226). CD169⁺ macrophages share the common macrophage markers except for F4/80, which is only expressed on some CD169⁺ macrophages (207). CD169 is expressed on marginal zone macrophages in the spleen, and subcapsular sinus macrophages in the lymph node (207). These macrophages line the barrier between draining blood or lymph and lymphocytes, and can capture draining antigens, suggesting they could play a critical role as vaccine targets. However, CD169⁺ macrophages themselves are poorly phagocytic; rather than presenting antigen, they shuttle intact antigens to B cell follicles to generate antibody responses (227, 228). Alternatively, CD169⁺ macrophages have been shown to transfer antigen to cDC1s to generate CD8⁺ T cell responses, suggesting they are important for all immune responses (229). Because of their selective expression in lymphoid tissues and role in coordinating both B and T cell responses, CD169⁺ macrophages may be a desirable target for targeted vaccines.

While targeting CD169⁺ macrophages is a new concept in vaccine design, a few studies have demonstrated the potential of this approach in mice. van Dinther et al. showed that targeting of OVA to CD169⁺ macrophages using anti-CD169 mAb-antigen conjugates resulted in anti-tumor CD8⁺ T cell responses and slowed progression of OVA-bearing tumors (230). Nijen Twilhaar et al. targeted CD169⁺ macrophages using the liposomes decorated with the ganglioside GM3, a natural ligand for CD169. GM3 liposomes loaded with OVA peptides were able to raise both CD8⁺ and CD4⁺ T cell responses (231). Edgar et al. instead targeted CD169⁺ macrophages with liposomes decorated with a synthetic glycan that binds CD169, termed CD169L (232, 233). CD169L liposomes loaded with OVA alone generated CD4⁺ T cell responses. However, when a mix of TLR7 agonist-loaded and OVA-loaded CD169L liposomes were used, both CD8⁺ and CD4⁺ T cell responses were generated, suggesting that CD169 targeting can be used to modulate CD8⁺ and CD4⁺ T cell ratios (233). In CD169^-/- mice, both T cell responses were diminished, indicating the dependence of these responses on CD169. Interestingly, Lisk et al. showed the antibody production of TLR adjuvant-based vaccines was diminished in CD169^-/- mice, suggesting the importance of CD169 in TLR-based vaccines (234). Overall, given the importance of CD169⁺ macrophages in shuttling antigen into lymph nodes, targeting them may represent a useful strategy in improving cellular and humoral responses to vaccines.

Neutrophils comprise most circulating granulocytes. They are recruited to sites of inflammation by chemokines and cytokines, where they release reactive oxygen species (ROS) and immunostimulatory molecules (238). They may also form neutrophil extracellular traps (NETs), web-like structures of DNA that trap and kill pathogens (241, 242). Neutrophils are typically identified by their expression of CD11b and Ly6G (Gr-1) (241, 242). Targeting of neutrophils has been widely studied for the treatment of inflammatory and autoimmune diseases rather than vaccination. Wang et al. and Zhang et al. showed that albumin nanoparticles can passively target activated neutrophils; however, this targeting effect was dependent on nanoparticle opsonization and inflammation-driven upregulation of FcγRII and FcγRIII on neutrophils (243, 244). Active targeting of neutrophils has been accomplished using both antibody and peptide ligands. Vij et al. used PLGA nanoparticles conjugated to an anti-Ly6G/6C antibody to deliver anti-inflammatory drugs to neutrophils (245). A limitation of this targeting strategy is that Ly6C is not specific to neutrophils and could also target other innate immune cells, such as monocytes and monocyte-derived cells. Cruz et al. developed a liposome which used Ly6G Fabs for neutrophil-specific targeting in order to deliver drugs that modulate neutrophil activity (246). Further specificity was conferred by adding a peptide derived from α1-antitrypsin, the natural ligand for neutrophil elastase, a surface protein exclusively expressed on activated neutrophils (246, 247). Volz et al. generated a neutrophil-specific targeting peptide by phage display which binds CD177, a neutrophil-specific activation marker. This CD177-targeting peptide was used to deliver PLGA nanoparticles to neutrophils containing immunomodulatory molecules (248).

For targeted vaccines, almost no studies have directly measured the response of targeting vaccines to neutrophils. It is known that alum based vaccines recruits neutrophils to injection sites and other large positively charged nanocarriers also induce neutrophil recruitment, which is especially helpful for generation of antibacterial Th1/Th17 responses (239, 240, 249, 250). It may be possible that neutrophils assist in this Th1/Th17 response, but further research is required to determine how targeting neutrophils effects vaccination efficacy.

Eosinophils, basophils, and mast cells comprise the remaining populations of granulocytes. They are generally involved in Th2 responses, and release immunostimulatory molecules upon activation. Eosinophils are activated by IL-5 in response to allergy or parasitic infections, while basophils and mast cells degranulate in response to crosslinking of surface-bound IgE and activation of the high-affinity Fc receptor for IgE (FcϵR1) (238). Eosinophils are typically identified by expression of CD11b, CCR3, and Siglec-F (mouse)/Siglec-8 (human) (251). Basophils and mast cells share the markers CD11b, IL3Ra (CD123), FcϵR1, and CD203c (ENPP-3), with basophils additionally expressing CD49b and mast cells additionally expressing c-kit (CD117) (252, 253).

Eosinophils, basophils, and mast cells have generally been targeted for the treatment of allergic diseases. Siglec-F/Siglec-8 is a common eosinophil target, and Siglec-8 antibody targeting for eosinophil depletion has been used in clinical trials to treat eosinophilic disorders (254). For targeted delivery to eosinophils, Nycholat et al. developed synthetic glycans that bind both Siglec-F/Siglec-8, and showed that liposomes decorated with these glycans effectively trafficked to eosinophils in vivo (255). In humans, basophils and mast cells express Siglec-3 (CD33) (256), and Duan et al. showed that liposomes decorated with a synthetic glycan targeting Siglec-3 were able to target human mast cells in a CD33-transgenic mouse model (257, 258). Targeting of basophils and mast cells can also be achieved through the exclusively expressed membrane-associated protein CD203c. Gold nanoparticles functionalized with anti-CD203c antibodies were shown to bind human basophil and mast cells in vitro; however, in vivo targeting has yet to be demonstrated with this system (259, 260). As with neutrophils, these granulocytes are also understudied for their participation in vaccination responses, particularly vaccines engineered for type 2 immune responses.

Despite sharing a name with DCs, follicular dendritic cells (FDCs) are a functionally distinct cell subset with a key role in adaptive immunity. FDCs arise from mesenchymal, rather than hematopoietic progenitors, only sharing the characteristic dendrite morphology (283, 284). FDCs are a rare population that reside in the B cell follicles, but do not process antigen. Instead, FDCs retain antigens in native form via immune complexes for presentation to germinal center B cells (283–286). B cells with high-affinity, antigen-specific BCRs bind retained antigen, and receive survival signals from FDCs, a critical process known as affinity maturation (283–286). Thus, vaccines targeting FDCs may be able to generate robust humoral responses.

FDCs are typically identified by expression of gp38 (PDPN) and CD21/35 (285, 286). Given their location in B cell follicles, targeting strategies that utilize opsonization and CD21/35 binding for B cell targeting may also be applicable to FDCs. Zhang et al. showed that both 15-50 nm-sized and 50-100 nm-sized antigen-conjugated gold nanoparticles were able to leverage lymphatic transport and traffic to lymph nodes (287). However, 50-100 nm-sized nanoparticles preferentially accumulated on FDCs due to increased C3b opsonization and CD21/35 binding (287). Mattsson et al. and Schussek et al. showed that a CD21/35-binding adjuvant derived from cholera toxin bound to FDCs, and strongly potentiated antibody and Tfh responses (288, 289). Aung et al. demonstrated that FDCs could be targeted with antigens fused to anti-CD35 ScFvs (290). After vaccination, targeted antigen was retained on FDCs longer, and significantly increased antigen-specific IgG, compared to a non-targeted control (290). Given the importance of FDCs to mature antibody production and humoral immune responses, further research on antigen targeting to FDCs is warranted.

LECs line the lymphatic vessels and lymph nodes. As a result, they are poised to interact with antigens and immune cells as they enter lymphatic circulation. LECs are capable of antigen capture and presentation to T cells (291–298). Under steady-state conditions, LECs are also known to generate long-lived memory CD8⁺ T cells that can differentiate into effector cells after re-challenge (294). Under inflammatory conditions such as viral infections, antigen persistence in LECs has been observed in a mechanism distinct from FDC retention (291–293). This persistence enhanced memory CD8⁺ T cell responses against infections. Thus, vaccines targeting LECs may be able to generate robust memory T cell responses.

LECs are typically identified by expression of gp38 (PDPN), CD31, LYVE1, and VEGFR3 (299, 300). While no LEC-targeting vaccine formulations have been reported, Guo et al. demonstrated that LEC-targeting magnetic nanoparticles for magnetic resonance imaging (MRI), functionalized with anti-LYVE-1 mAbs, were able to target LECs in vitro (301). Wu et al. further showed that magnetic nanoparticles functionalized with both anti-LYVE-1 and anti-gp38 mAbs were able to target LECs in vivo (302). While targeting systems exist, further research will also be required to show how selectively targeting vaccines to LECs affects adaptive responses.