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MHC class II (MHC II) Ag presentation by dendritic cells (DCs) is critical for CD4 + T cell immunity. Cell surface levels of MHC II loaded with peptide is controlled by ubiquitination. In this study, we have examined how MHC II ubiquitination impacts immunity using MHC IIKR KI/KI mice expressing mutant MHC II molecules that are unable to be ubiquitinated. Numbers of conventional DC (cDC) 1, cDC2 and plasmacytoid DCs were significantly reduced in MHC IIKR KI/KI spleen, with the remaining MHC IIKR KI/KI DCs expressing an altered surface phenotype. Whereas Ag uptake, endosomal pH, and cathepsin protease activity were unaltered, MHC IIKR KI/KI cDC1 produced increased inflammatory cytokines and possessed defects in Ag proteolysis. Immunization of MHC IIKR KI/KI mice identified impairments in MHC II and MHC class I presentation of soluble, cell-associated and/or DC-targeted OVA via mAb specific for DC surface receptor Clec9A (anti-Clec9A-OVA mAb). Reduced T cell responses and impaired CTL killing was observed in MHC IIKR KI/KI mice following immunization with cell-associated and anti-Clec9A-OVA. Immunization of MHC IIKR KI/KI mice failed to elicit follicular Th cell responses and generated barely detectable Ab to anti-Clec9A mAb-targeted Ag. In summary, MHC II ubiquitination in DCs impacts the homeostasis, phenotype, cytokine production, and Ag proteolysis by DCs with consequences for Ag presentation and T cell and Ab-mediated immunity., (Copyright © 2021 by The American Association of Immunologists, Inc.)

DEC-205 is a cell-surface receptor that transports bound ligands into the endocytic pathway for degradation or release within lysosomal endosomes. This receptor has been reported to bind a number of ligands, including keratin, and some classes of CpG oligodeoxynucleotides (ODN). In this study, we explore in detail the requirements for binding ODNs, revealing that DEC-205 efficiently binds single-stranded, phosphorothioated ODN of ≥14 bases, with preference for the DNA base thymidine, but with no requirement for a CpG motif. DEC-205 fails to bind double-stranded phosphodiester ODN, and thus does not bind the natural type of DNA found in mammals. The ODN binding preferences of DEC-205 result in strong binding of B class ODN, moderate binding to C class ODN, minimal binding to P class ODN, and no binding to A class ODN. Consistent with DEC-205 binding capacity, induction of serum IL-12p70 or activation of B cells by each class of ODN correlated with DEC-205 dependence in mice. Thus, the greater the DEC-205 binding capacity, the greater the dependence on DEC-205 for optimal responses. Finally, by covalently linking a B class ODN that efficiently binds DEC-205, to a P class ODN that shows poor binding, we improved DEC-205 binding and increased adjuvancy of the hybrid ODN. The hybrid ODN efficiently enhanced induction of effector CD8 T cells in a DEC-205-dependent manner. Furthermore, the hybrid ODN induced robust memory responses, and was particularly effective at promoting the development of liver tissue-resident memory T cells., (Copyright © 2021 by The American Association of Immunologists, Inc.)

Follicular dendritic cells and macrophages have been strongly implicated in presentation of native Ag to B cells. This property has also occasionally been attributed to conventional dendritic cells (cDC) but is generally masked by their essential role in T cell priming. cDC can be divided into two main subsets, cDC1 and cDC2, with recent evidence suggesting that cDC2 are primarily responsible for initiating B cell and T follicular helper responses. This conclusion is, however, at odds with evidence that targeting Ag to Clec9A (DNGR1), expressed by cDC1, induces strong humoral responses. In this study, we reveal that murine cDC1 interact extensively with B cells at the border of B cell follicles and, when Ag is targeted to Clec9A, can display native Ag for B cell activation. This leads to efficient induction of humoral immunity. Our findings indicate that surface display of native Ag on cDC with access to both T and B cells is key to efficient humoral vaccination., (Copyright © 2020 by The American Association of Immunologists, Inc.)

Targeting Ag to surface receptors on conventional type 1 dendritic cells can enhance induction of Ab and T cell responses. However, it is unclear to what extent the targeted receptor influences the resulting responses. In this study, we target Ag to Xcr1, Clec9A, or DEC-205, surface receptors that are expressed on conventional type 1 dendritic cells, and compare immune responses in BALB/c and C57BL/6 mice in vitro and in vivo after intradermal DNA vaccination. Targeting hemagglutinin from influenza A to Clec9A induced Ab responses with higher avidity that more efficiently neutralized influenza virus compared with Xcr1 and DEC-205 targeting. In contrast, targeting Xcr1 resulted in higher IFN-γ + CD8 + T cell responses in spleen and lung and stronger cytotoxicity. Both Clec9A and Xcr1 targeting induced Th1-polarized Ab responses, although the Th1 polarization of CD4 + T cells was more pronounced after Xcr1 targeting. Targeting DEC-205 resulted in poor Ab responses in BALB/c mice and a more mixed Th response. In an influenza challenge model, targeting either Xcr1 or Clec9A induced full and long-term protection against influenza infection, whereas only partial short-term protection was obtained when targeting DEC-205. In summary, the choice of targeting receptor, even on the same dendritic cell subpopulation, may strongly influence the resulting immune response, suggesting that different targeting strategies should be considered depending on the pathogen., (Copyright © 2020 by The American Association of Immunologists, Inc.)

CD4 + T cell responses are crucial for the control of many intracellular pathogens, yet the requirements for their induction are not fully understood. To better understand the role that various dendritic cell (DC) subtypes play in CD4 + T cell priming, we compared in vivo T cell responses to skin inoculation of mice with infectious or UV-inactivated HSV type 1. Localized infection elicited a Th1 response that was primed in skin-draining lymph nodes involving Ag presentation by migratory dermal and lymph node-resident DC. However, expansion and Th1 differentiation was impaired in response to UV-inactivated virus (UV-HSV), and this defect correlated with a restriction of Ag presentation to migratory CD103 - dermal DC. A similar differentiation defect was seen in infected mice lacking CD8α + and CD103 + classical type 1 DC (cDC1). Finally, Th1 differentiation after UV-HSV inoculation was rescued by targeted Ag delivery to CD8α + and CD103 + cDC1 using an anti-Clec9A Ab construct. This suggests that Ag presentation by cDC1 is crucial for optimal Th1 immunity to HSV type 1 infection and potentially other pathogens of the skin., (Copyright © 2019 by The American Association of Immunologists, Inc.)

Targeting Ags to dendritic cell (DC) surface receptors can induce a variety of responses depending on the DC type targeted, the receptor targeted, and the adjuvant used. Clec9A (DNGR-1), which is expressed by CD8(+) DCs, has been shown to bind F-actin exposed on damaged cells. Targeting Ag to this receptor in mice and nonhuman primates induces strong humoral immunity even in the absence of adjuvant, a process seen for a few select DC receptors. In contrast with other receptors, however, targeting Clec9A induces long-lived, affinity-matured Ab responses that are associated with efficient CD4(+) T cell responses shown to possess properties of follicular Th cells (TFH). In this article, we provide definitive evidence that Clec9A targeting promotes the development of TFH by showing that responding CD4 T cells express CXCR5, PD1, the TFH transcription factor Bcl6, and the cytokine IL-21, and that these cells localize to germinal centers. Furthermore, we extend studies from the model Ag OVA to the viral Ag glycoprotein D of HSV-1 and examine the capacity of primed TFH to form functional memory. We show that targeting glycoprotein D to Clec9A even in the absence of adjuvant induced long-lived memory CXCR5(+) PD1(hi) CD4(+) T cells that proliferated extensively upon secondary challenge and rapidly developed into effector TFH. This was associated with enhanced germinal center B cell responses and accelerated Ab production. Our study indicates that targeting Ags to Clec9A in the absence of adjuvant routinely generates TFH responses that form long-lived memory capable of robust secondary TFH responses., (Copyright © 2015 by The American Association of Immunologists, Inc.)

We established a humanized mouse model incorporating FLT3-ligand (FLT3-L) administration after hematopoietic cell reconstitution to investigate expansion, phenotype, and function of human dendritic cells (DC). FLT3-L increased numbers of human CD141(+) DC, CD1c(+) DC, and, to a lesser extent, plasmacytoid DC (pDC) in the blood, spleen, and bone marrow of humanized mice. CD1c(+) DC and CD141(+) DC subsets were expanded to a similar degree in blood and spleen, with a bias toward expansion of the CD1c(+) DC subset in the bone marrow. Importantly, the human DC subsets generated after FLT3-L treatment of humanized mice are phenotypically and functionally similar to their human blood counterparts. CD141(+) DC in humanized mice express C-type lectin-like receptor 9A, XCR1, CADM1, and TLR3 but lack TLR4 and TLR9. They are major producers of IFN-λ in response to polyinosinic-polycytidylic acid but are similar to CD1c(+) DC in their capacity to produce IL-12p70. Although all DC subsets in humanized mice are efficient at presenting peptide to CD8(+) T cells, CD141(+) DC are superior in their capacity to cross-present protein Ag to CD8(+) T cells following activation with polyinosinic-polycytidylic acid. CD141(+) DC can be targeted in vivo following injection of Abs against human DEC-205 or C-type lectin-like receptor 9A. This model provides a feasible and practical approach to dissect the function of human CD141(+) and CD1c(+) DC and evaluate adjuvants and DC-targeting strategies in vivo.

The response of B cells to Ag targeted to Clec9A on dendritic cells was followed using the hapten nitrophenol (NP) conjugated to rat Ig carrier. Injection of small amounts of NP conjugated to anti-Clec9A in the absence of adjuvants gave high and very prolonged Ab responses, approaching those obtained by high doses of nontargeted NP-protein conjugates with alum adjuvant. The response to NP-anti-Clec9A included the transient formation of germinal centers, maturation of Ab affinity, and some memory B cell formation. Serum Ab titers remained high 35 wk postimmunization, well after the initial follicular response had faded. The results suggest Clec9A-targeting strategies for improving Ab responses to vaccine Ags.

Three surface molecules of mouse CD8(+) dendritic cells (DCs), also found on the equivalent human DC subpopulation, were compared as targets for Ab-mediated delivery of Ags, a developing strategy for vaccination. For the production of cytotoxic T cells, DEC-205 and Clec9A, but not Clec12A, were effective targets, although only in the presence of adjuvants. For Ab production, however, Clec9A excelled as a target, even in the absence of adjuvant. Potent humoral immunity was a result of the highly specific expression of Clec9A on DCs, which allowed longer residence of targeting Abs in the bloodstream, prolonged DC Ag presentation, and extended CD4 T cell proliferation, all of which drove highly efficient development of follicular helper T cells. Because Clec9A shows a similar expression pattern on human DCs, it has particular promise as a target for vaccines of human application.

We have cloned the mouse and human C-type lectin Clec12A, expressed both, and produced mAb recognizing both. Mouse Clec12A is highly expressed on splenic CD8(+) dendritic cells (DC) and plasmacytoid DC. A proportion of CD8(-)DC also expresses lower levels of Clec12A, as do monocytes, macrophages, and B cells. Human CLEC12A, like the mouse counterpart, is expressed on blood monocytes and DC, including pDC and BDCA-3(+)DC, the proposed equivalent of mouse CD8(+)DC. To determine whether Ag targeted to Clec12A could induce immune responses, mice were injected with a rat mAb recognizing Clec12A, or a control rat mAb, then production of anti-rat Ig was measured. Anti-Clec12A mAb alone produced only moderate responses, but these were amplified by coinjecting only small amounts of LPS as a DC activation agent. Furthermore, when OVA was conjugated to anti-Clec12A mAb, OVA-specific T cells were induced to proliferate. This Ag presentation to naive T cells was due to targeting conventional DC, because their ablation eliminated T cell activation. The potent Ab responses induced using microgram amounts of anti-Clec12A and minimal amounts of adjuvant demonstrate that this molecule can be used as an Ag-delivery target to enhance Ab responses to vaccines.

A normalized subtracted gene expression library was generated from freshly isolated mouse dendritic cells (DC) of all subtypes, then used to construct cDNA microarrays. The gene expression profiles of the three splenic conventional DC (cDC) subsets were compared by microarray hybridization and two genes encoding signal regulatory protein beta (Sirpbeta1 and Sirpbeta4) molecules were identified as differentially expressed in CD8(-) cDC. Genomic sequence analysis revealed a third Sirpbeta member localized in the same gene cluster. These Sirpbeta genes encode cell surface molecules containing extracellular Ig domains and short intracytoplasmic domains that have a charged amino acid in the transmembrane region which can potentially interact with ITAM-bearing molecules to mediate signaling. Indeed, we demonstrated interactions between Sirpbeta1 and beta2 with the ITAM-bearing signaling molecule Dap12. Real-time PCR analysis showed that all three Sirpbeta genes were expressed by CD8(-) cDC, but not by CD8(+) cDC or plasmacytoid pre-DC. The related Sirpalpha gene showed a similar expression profile on cDC subtypes but was also expressed by plasmacytoid pre-DC. The differential expression of Sirpalpha and Sirpbeta1 molecules on DC was confirmed by staining with mAbs, including a new mAb recognizing Sirpbeta1. Cross-linking of Sirpbeta1 on DC resulted in a reduction in phagocytosis of Leishmania major parasites, but did not affect phagocytosis of latex beads, perhaps indicating that the regulation of phagocytosis by Sirpbeta1 is a ligand-dependent interaction. Thus, we postulate that the differential expression of these molecules may confer the ability to regulate the phagocytosis of particular ligands to CD8(-) cDC.

We demonstrate that functional and phenotypic equivalents of mouse splenic CD8(+) and CD8(-) conventional dendritic cell (cDC) subsets can be generated in vitro when bone marrow is cultured with fms-like tyrosine kinase 3 (flt3) ligand. In addition to CD45RA(high) plasmacytoid DC, two distinct CD24(high) and CD11b(high) cDC subsets were present, and these subsets showed equivalent properties to splenic CD8(+) and CD8(-) cDC, respectively, in the following: 1) surface expression of CD11b, CD24, and signal regulatory protein-alpha; 2) developmental dependence on, and mRNA expression of, IFN regulatory factor-8; 3) mRNA expression of TLRs and chemokine receptors; 4) production of IL-12 p40/70, IFN-alpha, MIP-1alpha, and RANTES in response to TLR ligands; 5) expression of cystatin C; and 6) cross-presentation of exogenous Ag to CD8 T cells. Furthermore, despite lacking surface CD8 expression, the CD24(high) subset contained CD8 mRNA and up-regulated surface expression when transferred into mice. This culture system allows access to bona fide counterparts of the splenic DC subsets.