The dendritic cells (DC) of mouse lymph nodes (LN) were isolated, analyzed for surface markers, and compared with those of spleen. Low to moderate staining of LN DC for CD4 and low staining for CD8 was shown to be attributable to pickup of these markers from T cells. Excluding this artifact, five LN DC subsets could be delineated. They included the three populations found in spleen (CD4+8−DEC-205−, CD4−8−DEC-205−, CD4−8+DEC-205+), although the CD4-expressing DC were of low incidence. LN DC included two additional populations, characterized by relatively low expression of CD8 but moderate or high expression of DEC-205. Both appeared among the DC migrating out of skin into LN, but only one was restricted to skin-draining LN and was identified as the mature form of epidermal Langerhans cells (LC). The putative LC-derived DC displayed the following properties: large size; high levels of class II MHC, which persisted to some extent even in CIITA null mice; expression of very high levels of DEC-205 and of CD40; expression of many myeloid surface markers; and no expression of CD4 and only low to moderate expression of CD8. The putative LC-derived DC among skin emigrants and in LN also showed strong intracellular staining of langerin.

Three distinct subtypes of dendritic cells (DC) are present in mouse spleen, separable as CD4−8α−, CD4+8α−, and CD4−8α+ DC. We have tested whether these represent stages of development or activation within one DC lineage, or whether they represent separate DC lineages. All three DC subtypes appear relatively mature by many criteria, but all retain a capacity to phagocytose particulate material in vivo. Although further maturation or activation could be induced by bacterially derived stimuli, phagocytic capacity was retained, and no DC subtype was converted to the other. Continuous elimination of CD4+8− DC by Ab depletion had no effect on the levels of the other DC subtypes. Bromodeoxyuridine labeling experiments indicated that all three DC subtypes have a rapid turnover (half-life, 1.5–2.9 days) in the spleen, with none being the precursor of another. The three DC subtypes showed different kinetics of development from bone marrow precursors. The CD8α+ spleen DC, apparently the most mature, displayed an extremely rapid turnover based on bromodeoxyuridine uptake and the fastest generation from bone marrow precursors. In conclusion, the three splenic DC subtypes behave as rapidly turning over products of three independent developmental streams.

The labeling kinetics of 5 dendritic cell (DC) subtypes within the lymphoid organs of healthy laboratory mice during continuous administration of bromodeoxyuridine (BrdU) was determined to investigate developmental relationships and determine turnover rates. Individual DC subtypes behaved as products of separate developmental streams, at least as far back as their dividing precursors. The rate of labeling varied with the lymphoid organ and the DC subtype. Labeling was faster overall in spleen and mesenteric lymph nodes (LNs) and slower in thymus and skin-draining LNs. The CD8+ DC subtype displayed the most rapid turnover, with a uniformly short (3-day) lifespan in spleen but with distinct short-lived and longer-lived subgroups in thymus. All the skin-derived DCs in LNs showed delayed and slow BrdU labeling, indicating a long overall lifespan; however, this was shown to reflect a long residence time in skin rather than a long-duration presenting antigen in the draining LN. Epidermal-derived Langerhans DCs displayed longer BrdU labeling lag and slower overall turnover than the dermal-derived DCs, and the movement of fluorescent Langerhans DC from skin to LN was slower than that of dermal DCs following skin painting with a fluorescent dye. However, once they arrived in lymphoid organs, all DCs present in healthy, uninfected mice displayed a rapid turnover, and this turnover was even faster after antigenic or microbial product stimulation.

Recognition of conserved features of infectious agents by innate pathogen receptors plays an important role in initiating the adaptive immune response. We have investigated early changes occurring among T cells after injection of TLR agonists into mice. Widespread, transient phenotypic activation of both naive and memory T cells was observed rapidly after injection of molecules acting through TLR3, -4, -7, and -9, but not TLR2. T cell activation was shown to be mediated by a combination of IFN-αβ, secreted by dendritic cells (DCs), and IFN-γ, secreted by NK cells; notably, IFN-γ-secreting NK cells expressed CD11c and copurified with DCs. Production of IFN-γ by NK cells could be stimulated by DCs from TLR agonist-injected mice, and although soluble factors secreted by LPS-stimulated DCs were sufficient to induce IFN-γ, maximal IFN-γ production required both direct contact of NK cells with DCs and DC-secreted cytokines. In vitro, IFN-αβ, IL-18, and IL-12 all contributed to DC stimulation of NK cell IFN-γ, whereas IFN-αβ was shown to be important for induction of T cell bystander activation and NK cell IFN-γ production in vivo. The results delineate a pathway involving innate immune mediators through which TLR agonists trigger bystander activation of T cells.

Follicular Th (TFH) cells have emerged as a new Th subset providing help to B cells and supporting their differentiation into long-lived plasma cells or memory B cells. Their differentiation had not yet been investigated following neonatal immunization, which elicits delayed and limited germinal center (GC) responses. We demonstrate that neonatal immunization induces CXCR5highPD-1high CD4+ TFH cells that exhibit TFH features (including Batf, Bcl6, c-Maf, ICOS, and IL-21 expression) and are able to migrate into the GCs. However, neonatal TFH cells fail to expand and to acquire a full-blown GC TFH phenotype, as reflected by a higher ratio of GC TFH/non-GC CD4+ T cells in immunized adults than neonates (3.8 × 10−3 versus 2.2 × 10−3, p = 0.01). Following the adoptive transfer of naive adult OT-II CD4+ T cells, OT-II TFH cells expand in the vaccine-draining lymph nodes of immunized adult but not infant recipients, whereas naive 2-wk-old CD4+ OT-II cells failed to expand in adult hosts, reflecting the influence of both environmental and T cell–intrinsic factors. Postponing immunization to later in life increases the number of TFH cells in a stepwise manner, in direct correlation with the numbers of GC B cells and plasma cells elicited. Remarkably, adjuvantation with CpG oligonucleotides markedly increased GC TFH and GC B cell neonatal responses, up to adult levels. To our knowledge, this is the first demonstration that the TFH cell development limits early life GC responses and that adjuvants/delivery systems supporting TFH differentiation may restore adultlike early life GC B cell responses.

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In the lung, dendritic cells (DC) are key antigen-presenting cells capable of triggering specific cellular responses to inhaled pathogens, and thus, they may be important in the initiation of an early response to mycobacterial infections. The ability of DC to enhance antigen presentation to naive T cells within the lungs was characterized with respect to Mycobacterium bovis Bacillus Calmette Guérin (BCG) vaccination against M. tuberculosis infection. In vitro derived DC were infected with BCG, which induced their maturation, as shown by the increased expression of MHC class II antigens, CD80 and CD86 co-stimulatory molecules. The synthesis of mRNA for IL-1, IL-6, IL-12, IL-10 and IL-1 receptor antagonist was also enhanced. When administered intratracheally in mice, infected DC induced a potent T cell response and the production of IFN-+ to mycobacterial antigens in the mediastinal lymph nodes, leading to a significant protection against aerosol M. tuberculosis infection. Intriguingly, although the vaccination schedule for BCG-infected DC was much shorter than subcutaneous BCG vaccination (7 days as compared to 100 days), both types of vaccination showed similar levels of protection. These data confirm that DC can be potent inducers of a cellular immune response against mycobacteria and support the concept of combining DC strategies with mycobacterial vaccines for protective immunity against tuberculosis. The first two authors contributed equally to this study.

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