Intestinal dendritic cells (DCs) continuously migrate through lymphatics to mesenteric lymph nodes where they initiate immunity or tolerance. Recent research has focused on populations of intestinal DCs expressing CD103. Here we demonstrate, for the first time, the presence of two distinct CD103(-) DC subsets in intestinal lymph. Similar to CD103(+) DCs, these intestine-derived CD103(-) DCs are responsive to Flt3 and they efficiently prime and confer a gut-homing phenotype to naive T cells. However, uniquely among intestinal DCs, CD103(-) CD11b(+) CX(3)CR1(int) lymph DCs induce the differentiation of both interferon-γ and interleukin-17-producing effector T cells, even in the absence of overt stimulation. Priming by CD103(-) CD11b(+) DCs represents a novel mechanism for the rapid generation of effector T-cell responses in the gut. Therefore, these cells may prove to be valuable targets for the treatment of intestinal inflammation or in the development of effective oral vaccines.

T follicular helper (Tfh) cells represent a recently defined CD4(+) T cell subset characterized by the expression of the chemokine receptor CXCR5 and an enhanced ability to support B cells to mount antibody responses. Here, we demonstrate that lymph-node-resident CXCR5(+) Tfh cells and gut-homing integrin alpha(4)beta(7)-expressing T helper cells are generated as separate subsets in the gut-draining mesenteric lymph nodes. Type I interferon signaling in dendritic cells and in nonhematopoietic cells selectively stimulates Tfh cell development in response to antigen in conjunction with Toll-like receptor (TLR)3 or TLR4 agonists. Consistent with this, the ability of dendritic cells to produce the cytokine IL-6, required for in vivo Tfh differentiation, and antibody affinity maturation are both reduced in absence of type I interferon signaling. Thus, our results identify type I interferon as a natural adjuvant that selectively supports the generation of lymph node resident Tfh cells.

T follicular helper (Tfh) cells are a subset of CD4 T cells that promote antibody production during vaccination. Conventional dendritic cells (cDCs) efficiently prime Tfh cells; however, conclusions regarding which cDC instructs Tfh cell differentiation have differed between recent studies. We found that these discrepancies might exist because of the unusual sites used for immunization in murine models, which differentially bias which DC subsets access antigen. We used intranasal immunization as a physiologically relevant route of exposure that delivers antigen to all tissue DC subsets. Using a combination of mice in which the function of individual DC subsets is impaired and different antigen formulations, we determined that CD11b migratory type 2 cDCs (cDC2s) are necessary and sufficient for Tfh induction. DC-specific deletion of the guanine nucleotide exchange factor DOCK8 resulted in an isolated loss of CD11b cDC2, but not CD103 cDC1, migration to lung-draining lymph nodes. Impaired cDC2 migration or development in DC-specific or knockout mice, respectively, led to reduced Tfh cell priming, whereas loss of CD103 cDC1s in mice did not. Loss of cDC2-dependent Tfh cell priming impaired antibody-mediated protection from live influenza virus challenge. We show that migratory cDC2s uniquely carry antigen into the subanatomic regions of the lymph node where Tfh cell priming occurs-the T-B border. This work identifies the DC subset responsible for Tfh cell-dependent antibody responses, particularly when antigen dose is limiting or is encountered at a mucosal site, which could ultimately inform the formulation and delivery of vaccines.

Mucosa-associated invariant T (MAIT) cells are innate-like T cells with a conserved TCR α-chain recognizing bacterial metabolites presented on the invariant MHC-related 1 molecule. MAIT cells are present in intestinal tissues and liver, and they rapidly secrete IFN-γ and IL-17 in response to bacterial insult. In colon cancer, IL-17–driven inflammation promotes tumor progression, whereas IFN-γ production is essential for antitumor immunity. Thus, tumor-associated MAIT cells may affect antitumor immune responses by their secreted cytokines. However, the knowledge of MAIT cell presence and function in tumors is virtually absent. In this study, we determined the frequency, phenotype, and functional capacity of MAIT cells in colon adenocarcinomas and unaffected colon lamina propria. Flow cytometric analyses showed significant accumulation of MAIT cells in tumor tissue, irrespective of tumor stage or localization. Colonic MAIT cells displayed an activated memory phenotype and expression of chemokine receptors CCR6 and CCR9. Most MAIT cells in unaffected colon tissues produced IFN-γ, whereas only few produced IL-17. Colonic MAIT cells also produced TNF-α, IL-2, and granzyme B. In the tumors, significantly lower frequencies of IFN-γ–producing MAIT cells were seen, whereas there were no differences in the other cytokines analyzed, and in vitro studies showed that secreted factors from tumor tissue reduced IFN-γ production from MAIT cells. In conclusion, MAIT cells infiltrate colon tumors but their ability to produce IFN-γ is substantially reduced. We suggest that MAIT cells have the capacity to promote local immune responses to tumors, but factors in the tumor microenvironment act to reduce MAIT cell IFN-γ production.

Salmonella typhimurium is a gram-negative bacterium that survives and replicates inside vacuolar compartments of macrophages. Infection of macrophages with S. typhimurium grown under conditions allowing expression of the type III secretion system results in apoptotic death of the infected cells. Here, we show that infection of bone marrow–derived macrophages (MΦ) with wild-type S. typhimurium 14028 results in presentation of epitopes derived from a bacteria-encoded antigen on major histocompatibility complex (MHC) class I and MHC class II molecules after internalization of apoptotic MΦ by bystander dendritic cells (DCs). In contrast, infection of MΦ with the phoP constitutive mutant strain CS022, which does not induce apoptosis in infected MΦ, does not result in presentation of a bacteria-derived antigen by bystander DCs unless the infected MΦ are induced to undergo apoptosis by treatment with lipopolysaccharide and ATP. DCs appear to be unique in their ability to present antigens derived from MΦ induced to undergo apoptosis by Salmonella, as bystander MΦ are not capable of presenting the bacteria-derived antigen despite the fact that they efficiently internalize the apoptotic cells. These data suggest that apoptosis induction by bacterial infection of MΦ may not be a quiescent death that allows the bacteria to escape recognition by the immune system, but rather may contribute to an antimicrobial immune response upon engulfment by bystander DCs.

The origins of dendritic cells (DCs) are poorly understood. In inflammation, DCs can arise from blood monocytes (MOs), but their steady-state origin may differ, as shown for Langerhans cells. Two main subsets of MOs, defined by expression of different chemokine receptors, CCR2 and CX3CR1, have been described in mice and humans. Recent studies have identified the inflammatory function of CCR2highCX3CR1low MOs but have not defined unambiguously the origin and fate of CCR2lowCX3CR1high cells. In this study, we show that rat MOs can also be divided into CCR2highCX3CR1low(CD43low) and CCR2lowCX3CR1high(CD43high) subsets with distinct migratory properties in vivo. Using whole body perfusion to obtain MOs, including the marginating pool, we show by adoptive transfer that CD43low MOs can differentiate into CD43high MOs in blood without cell division. By adoptive transfer of blood MOs followed by collection of pseudoafferent lymph, we show for the first time that a small proportion of intestinal lymph DCs are derived from CCR2lowCX3CR1high(CD43high) blood MOs in vivo under steady-state conditions. This study confirms one of the possible origins of CCR2lowCX3CR1high blood MOs and indicate that they may contribute to migratory intestinal DCs in vivo in the absence of inflammatory stimuli.

This study examines innate immunity to oral Salmonella during primary infection and after secondary challenge of immune mice. Splenic NK and NKT cells plummeted early after primary infection, while neutrophils and macrophages (Mφ) increased 10- and 3-fold, respectively. In contrast, immune animals had only a modest reduction in NK cells, no loss of NKT cells, and a slight increase in phagocytes following secondary challenge. During primary infection, the dominant sources of IFN-γ were, unexpectedly, neutrophils and Mφ, the former having intracellular stores of IFN-γ that were released during infection. IFN-γ-producing phagocytes greatly outnumbered IFN-γ-producing NK cells, NKT cells, and T cells during the primary response. TNF-α production was also dominated by neutrophils and Mφ, which vastly outnumbered NKT cells producing this cytokine. Neither T cells nor NK cells produced TNF-α early during primary infection. The TNF-α response was reduced in a secondary response, but remained dominated by neutrophils and Mφ. Moreover, no significant IFN-γ production by Mφ was associated with the secondary response. Indeed, only NK1.1+ cells and T cells produced IFN-γ in these mice. These studies provide a coherent view of innate immunity to oral Salmonella infection, reveal novel sources of IFN-γ, and demonstrate that immune status influences the nature of the innate response.

Dendritic cells (DC) present peripheral Ags to T cells in lymph nodes, but also influence their differentiation (tolerance/immunity, Th1/Th2). To investigate how peripheral conditions affect DC properties and might subsequently regulate T cell differentiation, we examined the effects of a potent DC-activating, TLR-4-mediated stimulus, LPS, on rat intestinal and hepatic DC in vivo. Steady-state rat intestinal and hepatic lymph DC are αE2 integrinhigh (CD103) and include two subsets, signal regulatory protein α (SIRPα)hi/low, probably representing murine CD8αα−/+ DC. Steady-state lamina propria DC are immature; surface MHC class IIlow, but steady-state lymph DC are semimature, MHC class IIhigh, but CD80/86low. Intravenous LPS induced rapid lamina propria DC emigration and increased lymph DC traffic without altering SIRPαhigh/SIRPαlow proportions. CD80/86 expression on lymph or mesenteric node DC was not up-regulated after i.v. LPS. In contrast, i.v. LPS stimulated marked CD80/86 up-regulation on splenic DC. CD80/86 expression on intestinal lymph DC, however, was increased after in vitro culture with TNF-α or GM-CSF, but not with up to 5 μg/ml LPS. Steady-state SIRPαlow DC localized to T cell areas of mesenteric nodes, spleen, and Peyer’s patch, whereas SIRPαhigh DC were excluded from these areas. Intravenous LPS stimulated rapid and abundant SIRPαhigh DC accumulation in T cell areas of mesenteric nodes and spleen. In striking contrast, i.v. LPS had no effect on DC numbers or distribution in Peyer’s patches. Our results suggest that any explanation of switching between tolerance and immunity as well as involving changes in DC activation status must also take into account differential migration of DC subsets.