Vaccination is a crucial method for inducing protective immunity, but its effectiveness can be limited in infants. The newborn immune system differs significantly from the adult system, both quantitatively and qualitatively, resulting in weakened immune responses in early life. This is partly a necessary adaptation to tolerate the sudden exposure to commensal microbes and environmental antigens. However, these differences also increase infants' susceptibility to infections. Adaptive immune responses and protective immunity rely on antigen presentation and T-cell priming. Therefore, improving T-cell priming by designing vaccines tailored to the age-specific immune parameters of neonates and infants is a promising strategy to enhance vaccination efficacy and immunity in early life. Conventional dendritic cells (cDCs) are powerful activators of naive T cells successfully used to induce adaptive immune responses in adults. However, in early life, these cells are often considered underdeveloped or functionally immature. In both murine and human neonates, the cDC compartment is smaller than in adults, and neonatal cDCs express lower levels of MHCII and costimulatory molecules, making them less effective at stimulating T cells. The early life murine cDC compartment also shows a bias towards Th2 responses due to delayed IL-12p70 production. A similar Th2 bias is seen in cord blood cDCs. Nevertheless, expanding cDCs in early life through FLT3L administration improves innate and adaptive immune defense in mice, suggesting that early-life cDCs possess the potential to initiate functional immune responses. The cDC compartment comprises distinct subsets with unique functions. cDC1 are potent cross-presenters and activates CD8+ T cells, promoting Th1 differentiation. cDC2 are more potent activators of CD4+ T cells and induce Th2, Th17, and T follicular helper cell differentiation. Neonatal mice show a predominance of cDC1, while cDC2 are the main cDC subtype in most adult organs. The Th2 bias in early life isn't solely explained by subset distribution because the relative frequency of cDC1 and cDC2 remains relatively stable throughout life in most human tissues. This suggests additional age-dependent regulation of cDC function. Neonatal cDC1, for instance, are less responsive to IFN-α signaling, produce less IL-12 but more IL-10, and have a reduced ability to activate CD4+ T cells compared to adult cDCs. Studies on cDC2 in early life are limited, but evidence suggests reduced IFN-γ production and impaired T cell activation compared to adult counterparts. Despite this, they can still promote Th2-mediated allergy and induce some CD8+ T cell proliferation under certain conditions. This raises the question of whether their T cell activation capacity can be harnessed for broader application, such as initiating T cell responses in the spleen, a major site for antibody production. The initial blood and immune cells originate from extra-embryonic yolk sac progenitors. For some cell types, like macrophages and mast cells, these yolk sac-derived cells persist after birth. cDCs arise during embryogenesis, contributing significantly to fetomaternal tolerance, but their origins in early life remain unclear. In adults, cDCs develop from bone marrow progenitors, with DNGR-1 expression distinguishing cDC-restricted progenitors. This study aims to investigate the differences between cDC2 in early and adult life and identify the mechanisms underlying age-dependent functional regulation. The authors hypothesize that cDC2 development is regulated in waves, leading to functionally competent cells in early life that respond differently to pathogens compared to adult cDCs.

The existing literature highlights the significant differences between the neonatal and adult immune systems, particularly concerning dendritic cell function. Studies have shown that neonatal dendritic cells exhibit a smaller compartment size, reduced expression of MHCII and costimulatory molecules, and an intrinsic bias towards Th2 responses. While the impact of these differences on vaccine efficacy is well-documented, the underlying mechanisms remain less understood. Previous research has established the distinct developmental pathways and functional roles of cDC1 and cDC2 subsets in adult immunity. However, the ontogeny and functional characteristics of cDC2 during early life have received comparatively less attention. This study builds upon previous work demonstrating the potential of early-life cDCs to initiate functional immune responses when stimulated by FLT3L, but delves deeper into the developmental origins and transcriptional regulation of cDC2 throughout life. This review also examines prior research on the ontogeny of other immune cell types, which shows that many develop in waves from distinct hematopoietic sources during development, suggesting that a similar pattern might be observed for cDC2. Studies examining the impact of cytokine environments and pathogen sensing on cDC function also informed the hypothesis of the current research.

This study employed a multifaceted approach using various techniques to comprehensively investigate the development and function of cDC2 in young and adult mice. The researchers utilized several mouse models, including *Clec9acre⁺/⁺Rosa*ᵀᴼᴹ mice, which express CRE-recombinase under the Clec9a promoter to track cells of the cDC lineage. *Clec9acre⁺/⁺Rosa*ᵀᴼᴹ mice, *Clec9acre/*creRosa^TOM mice, and *Clec9acre/*creRosa^YFP mice were used for fate mapping experiments to trace the origins of cDC2. Additional fate mapping experiments involved using *Flt3l*^−/−, *Csf1r*-Mer-iCre-MerRosa^YFP, *Myb*^-/-, *Rag1cre Rosa YFP*, and *Il7rcre Rosa RFP* mice. Flow cytometry was extensively used to identify and characterize cDC subsets based on surface markers such as CD11c, MHCII, CD24, CD11b, XCR1, and ESAM. To investigate transcriptional differences, the researchers performed bulk RNA sequencing on sorted TOM+ and TOM- cDC2 populations from young and adult mice. Single-cell RNA sequencing (scRNA-seq) was also performed on splenic MHCII+ cells from young mice to provide a more detailed analysis of the transcriptional landscape of cDC2 and their relationship with other immune cells. In vitro T-cell proliferation assays were conducted to assess the ability of cDC2 from different ages to activate and polarize naive T cells. OVA peptide 323–339 was used to pulse cDC2, which were then co-cultured with naive OT-II transgenic T cells in the presence or absence of T-cell polarizing cytokines. The production of cytokines like IFN-γ, IL-4, IL-17A, and Foxp3 was measured to assess T-cell differentiation. In vivo antigen targeting experiments were conducted using an antibody directed against the C-type lectin receptor CLEC4A4/DCIR2 to specifically deliver OVA to cDC2. The effect of CpG-B, a TLR9 agonist, was also investigated on cytokine production and T-cell activation. Statistical analyses, including two-tailed t-tests, paired t-tests, and one-way ANOVAs, were used to compare the results across different groups. Immunofluorescence microscopy was used to visualize the localization of cDC subsets in the spleen. In vitro phagocytosis assays were conducted to determine the ability of cDC2 from different age groups to take up fluorescently labeled latex beads.

This study revealed several key findings regarding the development and function of cDC2 in young and adult mice. Firstly, the authors demonstrated that early-life cDC2 exhibit ontogenetic diversity, originating from both Clec9a-positive and Clec9a-negative progenitors. The proportion of cDC2 derived from Clec9a-positive progenitors increases with age, suggesting a developmental shift in cDC2 origins. Fate mapping experiments showed a contribution of lymphoid progenitors to early-life cDC2, with this contribution decreasing as the mice age. Despite their different developmental origins, ontogenetically distinct cDC2 in early life (TOM+ and TOM-) were transcriptionally and phenotypically similar, indicating that cell origin is not the primary determinant of cDC2 function in early life. Instead, the study suggests that the cytokine environment plays a major role in shaping cDC2 function. Comparison of TOM+ cDC2 from young and adult mice revealed significant differences in gene expression profiles. Adult cDC2 showed enrichment of genes involved in inflammatory responses and signaling pathways downstream of IFN-γ, TNF-α, IL-2, and IFN-α, suggesting distinct cytokine signaling in the adult environment. In contrast, young cDC2 exhibited higher expression of genes associated with cell cycle progression. Functional assays showed that early-life cDC2, despite their distinct ontogeny, induced similar T-cell proliferation as adult cDC2. However, they differed in their ability to induce T-cell differentiation. Early-life cDC2 induced higher levels of Th17 and Treg differentiation than adult cDC2, both in vitro and in vivo when antigen was targeted using an anti-DCIR2 antibody. Furthermore, stimulation with CpG-B revealed distinct cytokine profiles in cDC2 from young and adult mice, with young cDC2 producing higher levels of IL-6, IL-12p40, TNF-α, IL-10, and IL-27. These differences in cytokine production were further highlighted when antigen was targeted to cDC2 in the presence of CpG-B, with young cDC2 inducing increased Th1 and TNF-α responses. Single-cell RNA sequencing identified a distinct cluster of RORγt-expressing cDC2 present only in early life, suggesting a unique cDC subset with transient expression. Overall, these findings indicate that age-dependent differences in the cytokine environment are more influential in shaping cDC2 function than their developmental origins.

This study provides novel insights into the development and functional regulation of cDC2, challenging the traditional view that early-life cDCs are functionally immature. The findings demonstrate that while cDC2 in early life originate from diverse hematopoietic sources, their functional competence is not significantly impacted by their distinct ontogeny. Instead, environmental cues, specifically the cytokine milieu, appear to be the major drivers of functional differences between early-life and adult cDC2. The identification of a distinct RORγt-expressing cDC2 population only in early life warrants further investigation to understand its function and developmental significance. The observation that early-life cDC2 exhibit a heightened capacity for Th17 and Treg differentiation is particularly relevant considering the increased susceptibility of neonates to infections requiring these responses. The distinct cytokine responses to CpG-B stimulation suggest potential avenues for vaccine adjuvant design to improve immune responses in early life by targeting these age-specific differences. The ability to successfully target cDC2 in vivo opens new avenues for developing vaccines designed specifically for the neonatal immune system. This study makes a significant contribution to our understanding of the neonatal immune system and provides a rational basis for developing more effective vaccines and immunotherapies for infants.

This study demonstrates that while early-life cDC2 exhibit developmental heterogeneity, their function is primarily shaped by environmental signals, particularly cytokine profiles. Early-life cDC2 are functionally competent and induce distinct T-cell responses compared to their adult counterparts. The identification of a unique RORγt+ cDC2 population in early life and the distinct cytokine response to CpG-B highlight potential avenues for designing improved vaccination strategies targeting neonates. Future research should focus on dissecting the specific signaling pathways and epigenetic modifications responsible for age-dependent changes in cDC2 function and explore the therapeutic potential of harnessing early-life cDC2 for boosting immunity.

While the study provides a comprehensive analysis of cDC2 development and function, several limitations should be noted. The study primarily focuses on splenic cDC2, and findings may not be generalizable to other tissues. The use of mouse models may not perfectly recapitulate the complexities of human neonatal immunity. The study primarily investigates a specific time point in early life (2-2.5 weeks), and further studies are needed to establish the full developmental trajectory of cDC2 function. The analysis of the RORγt+ cDC2 population is limited and further research is necessary to understand its precise functional role and developmental relationship to other cDC subsets. The study's in vivo antigen targeting experiments used an adjuvant, therefore these experiments may not accurately reflect immune responses in the complete absence of an adjuvant. Future research should explore the role of other adjuvants that may differentially modulate the function of early-life cDC2. Finally, the study didn’t explore the impact of the microbiota on cDC2 function, despite some suggestions that this may play a role.