Ulf Yrlid

Relationships between Distinct Blood Monocyte Subsets and Migrating Intestinal Lymph Dendritic Cells In Vivo under Steady-State Conditions

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.

In Vivo Activation of Dendritic Cells and T Cells during Salmonella enterica Serovar Typhimurium Infection

ABSTRACT The present study was initiated to gain insight into the interaction between splenic dendritic cells (DC) and Salmonella enterica serovar Typhimurium in vivo. Splenic phagocytic cell populations associated with green fluorescent protein (GFP)-expressing bacteria and the bacterium-specific T-cell response were evaluated in mice given S. enterica serovar Typhimurium expressing GFP and ovalbumin. Flow cytometry analysis revealed that GFP-positive splenic DC (CD11c+ major histocompatibility complex class II-positive [MHC-II+] cells) were present following bacterial administration, and confocal microscopy showed that GFP-expressing bacteria were contained within CD11c+MHC-II+ splenocytes. Furthermore, splenic DC and T cells were activated following Salmonella infection. This was shown by increased surface expression of CD86 and CD40 on CD11c+ MHC-II+ cells and increased CD44 and CD69 expression on CD4+ and CD8+ T cells.Salmonella-specific gamma interferon (IFN-γ)-producing cells in both of these T-cell subsets, as well as cytolytic effector cells, were also generated in mice given live bacteria. The frequency of Salmonella-specific CD4+ T cells producing IFN-γ was greater than that of specific CD8+ T cells producing IFN-γ in the same infected animal. This supports the argument that the predominant source of IFN-γ production by cells of the specific immune response is CD4+ T cells. Finally, DC that phagocytosed live or heat-killed Salmonella in vitro primed bacterium-specific IFN-γ-producing CD4+ and CD8+ T cells as well as cytolytic effector cells following administration into naı̈ve mice. Together these data suggest that DC are involved in priming naı̈ve T cells toSalmonella in vivo.

Intestinal Dendritic Cell Subsets: Differential Effects of Systemic TLR4 Stimulation on Migratory Fate and Activation In Vivo

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.

Subsets of migrating intestinal dendritic cells

Dendritic cells (DCs) in the intestine are heterogeneous. Phenotypically different populations of conventional DCs have been identified in the intestinal lamina propria, Peyer’s patches, and in the draining mesenteric lymph nodes, to which these DCs constitutively migrate. Markers used to identify these populations include major histocompatibility complex class II, CD11c, CD8α, CD11b, and CD103. Extensive studies in rats, summarized here, which involved collection of migrating DCs by thoracic duct cannulation after mesenteric lymphadenectomy, have clearly demonstrated that the subsets of migrating intestinal lymph DCs have different functional properties. The subsets might play different roles in the induction of oral tolerance and in driving systemic immune responses after vaccination or intestinal stimulation with Toll‐like receptor ligands. The use of these surgical techniques allows investigation of the functions of purified subsets of migrating DCs. However, in the rat, these studies are limited by the range of available reagents and are difficult to compare with data from other species in this fast‐moving field. Recent refinements have enabled the collection of migrating intestinal DCs from mice; our initial results are described here. We believe that these studies will generate exciting data and have the potential to resolve important questions about the functions of migrating intestinal DC subsets.

Regulation of Intestinal Dendritic Cell Migration and Activation by Plasmacytoid Dendritic Cells, TNF-α and Type 1 IFNs after Feeding a TLR7/8 Ligand

Dendritic cells (DCs) migrating via lymph are the primary influence regulating naive T cell differentiation, be it active immunity or tolerance. How DCs achieve this regulation in vivo is poorly understood. Intestinal DCs are in direct contact with harmless or pathogenic luminal contents, but may also be influenced by signals from epithelial cells, macrophages, or other resident or immigrant cells. To understand the role of TLR7 and TLR8 in regulating intestinal DC function, we fed a TLR7/8 ligand (resiquimod (R-848)) to rats and mice and examined DC in pseudoafferent lymph (rat) and mesenteric lymph nodes (MLNs). Oral R-848 induced a 20- to 30-fold increase in DC output from the intestine within 10 h due to a virtually total release of lamina propria DCs. This resulted in an accumulation of DCs in the MLNs that in mice was completely TNF-α dependent. Surprisingly, intestinal lymph DCs (iL-DCs) released by R-848 did not up-regulate CD86, but did up-regulate CD25. In contrast, MLN-DCs from R-848-stimulated rats and mice expressed high levels of CD86. This DC activation in MLNs was dependent on type 1 IFNs. The major source of these rapidly released cytokines is plasmacytoid DCs (pDCs) and not classical DCs, because depletion of pDCs significantly reduces the R-848-stimulated increase in serum cytokine levels as well as the accumulation and activation of DCs in MLNs. These experiments show that TLR-mediated regulation of iL-DC functions in vivo is complex and does not depend only on direct iL-DC stimulation, but can be regulated by pDCs.

CD11c(high )dendritic cells are essential for activation of CD4+ T cells and generation of specific antibodies following mucosal immunization.

To generate vaccines that protect mucosal surfaces, a better understanding of the cells required in vivo for activation of the adaptive immune response following mucosal immunization is required. CD11chigh conventional dendritic cells (cDCs) have been shown to be necessary for activation of naive CD8+ T cells in vivo, but the role of cDCs in CD4+ T cell activation is still unclear, especially at mucosal surfaces. The activation of naive Ag-specific CD4+ T cells and the generation of Abs following mucosal administration of Ag with or without the potent mucosal adjuvant cholera toxin were therefore analyzed in mice depleted of CD11chigh cDCs. Our results show that cDCs are absolutely required for activation of CD4+ T cells after oral and nasal immunization. Ag-specific IgG titers in serum, as well as Ag-specific intestinal IgA, were completely abrogated after feeding mice OVA and cholera toxin. However, giving a very high dose of Ag, 30-fold more than required to detect T cell proliferation, to cDC-ablated mice resulted in proliferation of Ag-specific CD4+ T cells. This proliferation was not inhibited by additional depletion of plasmacytoid DCs or in cDC-depleted mice whose B cells were MHC-II deficient. This study therefore demonstrates that cDCs are required for successful mucosal immunization, unless a very high dose of Ag is administered.

Plasmacytoid Dendritic Cells Do Not Migrate in Intestinal or Hepatic Lymph

Plasmacytoid dendritic cells (pDCs) recognize pathogen-associated molecules, particularly viral, and represent an important mechanism in innate defense. They may however, also have roles in steady-state tolerogenic responses at mucosal sites. pDCs can be isolated from blood, mucosa, and lymph nodes (LNs). Although pDCs can express peripherally derived Ags in LNs and at mucosal sites, it is not clear whether pDCs actually migrate from the periphery in lymph or whether LN pDCs acquire Ags by other mechanisms. To determine whether pDCs migrate in lymph, intestine or liver-draining LNs were removed and thoracic duct leukocytes (TDLs) were collected. TDLs expressing MHC-II and CD45R, but not TCRαβ or CD45RA, were then analyzed. These enriched TDLs neither transcribe type I IFNs nor secrete inflammatory cytokines in response to viral stimuli in vitro or after a TLR7/8 stimulus in vivo. In addition, these TDLs do not express CD5, CD90, CD200, or Siglec-H, but do express Ig, and therefore represent B cells, despite their lack of CD45RA expression. Intestinal and hepatic lymph are hence devoid of bona fide pDCs under both steady-state conditions and after TLR7/8 stimulation. This shows that any role for pDCs in Ag-specific T cell activation or tolerance must differ from the roles of classical dendritic cells, because it cannot result from peripheral Ag capture, followed by migration of pDCs via lymph to the LN.

Directed antigen targeting in vivo identifies a role for CD103(+) dendritic cells in both tolerogenic and immunogenic T-cell responses.

The αE integrin chain CD103 identifies a subset of migratory dendritic cells (DCs) in the gut, lung, and skin. To gain further understanding of the function of CD103+ DCs in regulating adaptive immunity in vivo, we coupled ovalbumin (OVA) to the CD103 antibody M290 (M290.OVA). Intraperitoneal injection of M290.OVA induced OVA-specific CD8+ and CD4+ T-cell proliferation in lymph nodes (LNs) of wild-type but not CD103−/− mice, or in mice depleted of CD11c+ cells. In the absence of maturation stimuli, systemic antigen targeting to CD103+ DCs led to tolerance of CD8+ T cells, whereas coadministration of adjuvant induced cytotoxic T-lymphocyte (CTL) immunity and antibody production. Mucosal intratracheal application of M290.OVA also induced T-cell proliferation in mediastinal LNs, yet the functional outcome was tolerance that inhibited subsequent development of allergic airway inflammation and immunoglobulin E (IgE) responses to inhaled OVA. These findings identify antigen targeting to CD103+ DCs as a potential strategy to regulate immune responses in nonlymphoid mucosal tissues.

A distinct subset of intestinal dendritic cells responds selectively to oral TLR7/8 stimulation

The intestinal innate immune system continually interacts with commensal bacteria, thus oral vaccines should induce extra/alternative activation of DC, potentially through TLR. To examine this we collected intestinal lymph DC (iL‐DC) under steady‐state conditions and after feeding resiquimod (R‐848), a synthetic TLR7/8 ligand, which we showed induces complete emptying of gut DC into lymph. iL‐DC are heterogeneous with subset‐specific functions. In this study we determined the kinetics of iL‐DC subset release, activation and cytokine secretion induced by R‐848. We show that L‐DC comprise three distinct subsets (CD172ahigh, CD172aint and CD172alow) present with similar frequencies in intestinal but not hepatic lymph. No iL‐DC express TLR7 mRNA, and only CD172a+ iL‐DC express TLR8. However, after oral R‐848 administration, output of all three subsets increases dramatically. CD172ahigh DC release precedes that of CD172alow DC, and the increased frequency of CD25high iL‐DC is restricted to the two CD172a+ subsets. After feeding R‐848 only CD172ahigh iL‐DC secrete IL‐6 and IL‐12p40. However, CD172aint and CD172ahigh DC secrete similar but markedly lower amounts when stimulated in vitro. These results highlight the importance of in vivo approaches to assess adjuvant effects on DC and give novel insights into the subset‐specific effects of an oral TLR ligand on intestinal DC.

Human Mucosa-Associated Invariant T Cells Accumulate in Colon Adenocarcinomas but Produce Reduced Amounts of IFN-γ

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.