T cell dynamics during induction of tolerance and suppression of experimental allergic encephalomyelitis

The cell dynamics associated with induction of peripheral T cell tolerance remain largely undefined. In this study, an in vivo model was adapted to two-photon microscopy imaging, and T cell behavior was analyzed on tolerogen-induced modulation. FcγR-deficient (FcγR(-/-)) mice were unable to resist or alleviate experimental allergic encephalomyelitis when treated with Ig-myelin oligodendrocyte glycoprotein (MOG) tolerogen, an Ig carrying the MOG35-55 peptide. However, when FcγR(+/+) dendritic cells (DCs) are adoptively transferred into FcγR(-/-) mice, uptake and presentation of Ig-MOG occurs and the animals were able to overcome experimental allergic encephalomyelitis. We then fluorescently labeled FcγR(+/+) DCs and 2D2 MOG-specific TCR-transgenic T cells, transferred them into FcγR(-/-) mice, administered Ig-MOG, and analyzed both T cell-DC contact events and T cell motility. The results indicate that tolerance takes place in lymphoid organs, and surprisingly, the T cells do not become anergic but instead have a Th2 phenotype. The tolerant Th2 cells displayed reduced motility after tolerogen exposure similar to Th1 cells after immunization. However, the Th2 cells had higher migration speeds and took longer to exhibit changes in motility. Therefore, both Th1 immunity and Th2 tolerance alter T cell migration on Ag recognition, but the kinetics of this effect differ among the subsets.     

Extracellular acidosis triggers the maturation of human dendritic cells and the production of IL-12

Although the development of an acidic tissue environment or acidosis is a hallmark of inflammatory processes, few studies analyze the effect of extracellular pH on immune cells. We have previously shown that exposure of murine dendritic cells (DCs) to pH 6.5 stimulates macropinocytosis and cross-presentation of extracellular Ags by MHC class I molecules. We report that the transient exposure of human DCs to pH 6.5 markedly increases the expression of HLA-DR, CD40, CD80, CD86, CD83, and CCR7 and improves the T cell priming ability of DCs. Incubation of DCs at pH 6.5 results in the activation of the PI3K/Akt and the MAPK pathways. Using specific inhibitors, we show that the maturation of DCs induced by acidosis was strictly dependent on the activation of p38 MAPK. DC exposure to pH 6.5 also induces a dramatic increase in their production of IL-12, stimulating the synthesis of IFN-gamma, but not IL-4, by Ag-specific CD4(+) T cells. Interestingly, we find that suboptimal doses of LPS abrogated the ability of pH 6.5 to induce DC maturation, suggesting a cross-talk between the activation pathways triggered by LPS and extracellular protons in DCs. We conclude that extracellular acidosis in peripheral tissues may contribute to the initiation of adaptive immune responses by DCs, favoring the development of Th1 immunity.     

Dendritic cell differentiation state and their interaction with NKT cells determine Th1/Th2 differentiation in the murine model of Leishmania major infection

Recent reports demonstrated that dendritic cells (DC) sense inflammatory and microbial signals differently, redefining their classical subdivision into an immature endocytic and a mature Ag-presenting differentiation stage. Although both signals induce DC maturation by up-regulating MHC class II and costimulatory molecules, only TLR signals such as LPS are able to trigger proinflammatory cytokine secretion by DCs, including Th1-polarizing IL-12. Here, we explored the murine Leishmania major infection model to examine the CD4(+) T cell response induced by differentially matured DCs. When partially matured TNF-DCs were injected into BALB/c mice before infection, the mice failed to control L. major infection and developed a Th2 response which was dependent on IL-4Ralpha signaling. In contrast, injections of fully matured LPS+CD40-DCs induced a Th1 response controlling the infection. Pulsing DCs with a lysate of L. major did not affect DC maturation with TNF-alpha or LPS+anti-CD40. When the expression of different Notch ligands on DCs was analyzed, we found increased expression of Th2-promoting Jagged2 in TNF-DCs, whereas LPS+CD40-DCs up-regulated the Th1-inducing Delta4 and Jagged1 molecules. The Th2 polarization induced by TNF-DCs required interaction with CD1d-restricted NKT cells. However, NKT cell activation by L. major lysate-pulsed DCs was not affected by blockade of the endogenous glycolipid, suggesting exchange with exogenous parasite-derived CD1 glycolipid Ag. In sum, the differentiation stage of DCs as well as their interaction with NKT cells determines Th1/Th2 differentiation. These results have generic implications for the understanding of DC-driven Th cell responses and the development of improved DC vaccines against leishmaniasis.     

H2-O expression in primary dendritic cells

H2-O is a nonpolymorphic class II molecule whose biological role remains to be determined. H2-O modulates H2-M function, and it has been generally believed to be expressed only in B lymphocytes and thymic medullary epithelial cells, but not in dendritic cells (DCs). In this study, we report identification of H2-O expression in primary murine DCs. Similar to B cells, H2-O is associated with H2-M in DCs, and its expression is differentially regulated in DC subsets as well as during cell maturation and activation. Primary bone marrow DCs and plasmacytoid DCs in the spleen and lymph nodes express MHC class II and H2-M, but not the inhibitor H2-O. In contrast, myeloid DCs in secondary lymphoid organs express both H2-M and H2-O. In CD8alphaalpha(+) DCs, the ratio of H2-O to H2-M is higher than in CD8alphaalpha(-) DCs. In DCs generated from GM-CSF- and IL-4-conditioned bone marrow cultures, H2-O expression is not detected regardless of the maturation status of the cells. Administration of LPS induces in vivo activation of myeloid DCs, and this activation is associated with down-regulation of H2-O expression. Primary splenic DCs from H2-O(-/-) and H2-O(+/+) mice present exogenous protein Ags to T cell hybridomas similarly well, but H2-O(-/-) DCs induce stronger allogeneic CD4 T cell response than the H2-O(+/+) DCs in mixed leukocyte reactions. Our results suggest that H2-O has a broader role than previously appreciated in regulating Ag presentation.     

Host dendritic cells alone are sufficient to initiate acute graft-versus-host disease

Alloantigen expression on host APCs is essential to initiate graft-vs-host disease (GVHD); however, critical APC subset remains to be elucidated. We compared the ability of dendritic cells (DCs) and B cells to initiate acute GVHD by an add-back study of MHC class II-expressing APCs (II(+/+)) into MHC class II-deficient (II(-/-)) mice that were resistant to CD4-dependent GVHD. Injection of host-derived, but not donor-derived, II(+/+) DCs or host-derived II(+/+) B cells, was sufficient to break GVHD resistance of II(-/-) mice and induced lethal acute GVHD. By contrast, host-derived II(+/+) B cells, both naive and LPS stimulated, failed to induce activation or tolerance of donor CD4(+) T cells. Similarly, in a model of CD8-dependent GVHD across MHC class I mismatch injection of allogeneic DCs, but not B cells, induced robust proliferation of donor CD8(+) T cells and broke GVHD resistance of chimeric recipients in which APCs were syngeneic to donors. These results demonstrate that host-derived DCs are critical in priming donor CD4(+) and CD8(+) T cells to cause GVHD, and selective targeting of host DCs may be a promising strategy to prevent GVHD.     

CD47 expression on T cell is a self-control negative regulator of type 1 immune response

The cytokine milieu and dendritic cells (DCs) direct Th1 development. Yet, the control of Th1 polarization by T cell surface molecules remains ill-defined. We here report that CD47 expression on T cells serves as a self-control mechanism to negatively regulate type 1 cellular and humoral immune responses in vivo. Th2-prone BALB/c mice that lack CD47 (CD47(-/-)) displayed a Th1-biased Ab profile at steady state and after immunization with soluble Ag. CD47(-/-) mice mounted a T cell-mediated exacerbated and sustained contact hypersensitivity (CHS) response. After their adoptive transfer to naive CD47-deficient hosts 1 day before immunization with soluble Ag, CD47(-/-) as compared with CD47(+/+)CD4(+) transgenic (Tg) T cells promoted the deviation of Ag-specific T cell responses toward Th1 that were characterized by a high IFN-gamma:IL-4 cytokine ratio. Although selective CD47 deficiency on DCs led to increased IL-12p70 production, CD47(-/-)Tg T cells produced more IFN-gamma and displayed higher T-bet expression than CD47(+/+) Tg T cells in response to OVA-loaded CD47(-/-) DCs. CD47 as part of the host environment has no major contribution to the Th1 polarization responses. We thus identify the CD47 molecule as a T cell-negative regulator of type 1 responses that may limit unwanted collateral damage to maximize protection and minimize host injury.     

Maturing dendritic cells depend on RAGE for in vivo homing to lymph nodes

The mobilization of dendritic cells (DCs) from peripheral tissues is critical for the establishment of T cell-dependent immune responses or tolerance, because the physical interaction of DCs with naive T cells takes place in the T cell areas of lymph nodes. The autocrine/paracrine release of the high mobility group box 1 (HMGB1) nuclear protein by DCs controls the outcome of the DC-T cell interaction, influencing the priming/Th1 polarization of naive T cells. We herein present evidence that the receptor for advanced glycation end products (RAGE), a multiligand member of the Ig superfamily of cell-surface molecules that acts as a receptor for HMGB1, plays a nonredundant role in DC homing to lymph nodes. We used noninvasive imaging by magnetic resonance and immunohistochemistry to track DCs after s.c. injection in the footpad of wild-type(+/+) or RAGE(-/-) mice. Maturing DCs expressing RAGE effectively migrated in both conditions. In contrast, RAGE(-/-) DCs failed to reach the draining popliteal lymph nodes of +/+ and -/- mice, indicating that the integrity of RAGE is required for DC mobilization. Thus the HMGB1-RAGE pathway is a checkpoint in DC maturation and function and a candidate for targeted therapies.     

Endotoxin-induced maturation of MyD88-deficient dendritic cells

LPS, a major component of the cell wall of Gram-negative bacteria, can induce a variety of biological responses including cytokine production from macrophages, B cell proliferation, and endotoxin shock. All of them were completely abolished in MyD88-deficient mice, indicating the essential role of MyD88 in LPS signaling. However, MyD88-deficient cells still show activation of NF-kappaB and mitogen-activated protein kinase cascades, although the biological significance of this activation is not clear. In this study, we have examined the effects of LPS on dendritic cells (DCs) from wild-type and several mutant mice. LPS-induced cytokine production from DCs was dependent on MyD88. However, LPS could induce functional maturation of MyD88-deficient DCs, including up-regulation of costimulatory molecules and enhancement of APC activity. MyD88-deficient DCs could not mature in response to bacterial DNA, the ligand for Toll-like receptor (TLR)9, indicating that MyD88 is differentially required for TLR family signaling. MyD88-dependent and -independent pathways originate at the intracytoplasmic region of TLR4, because both cytokine induction and functional maturation were abolished in DCs from C3H/HeJ mice carrying the point mutation in the region. Finally, in vivo analysis revealed that MyD88-, but not TLR4-, deficient splenic CD11c(+) DCs could up-regulate their costimulatory molecule expression in response to LPS. Collectively, the present study provides the first evidence that the MyD88-independent pathway downstream of TLR4 can lead to functional DC maturation, which is critical for a link between innate and adaptive immunity.     

High mobility group box protein 1: an endogenous signal for dendritic cell maturation and Th1 polarization

High mobility group box protein 1 (HMGB1), a DNA binding nuclear and cytosolic protein, is a proinflammatory cytokine released by monocytes and macrophages. This study addressed the hypothesis that HMGB1 is an immunostimulatory signal that induces dendritic cell (DC) maturation. We show that HMGB1, via its B box domain, induced phenotypic maturation of DCs, as evidenced by increased CD83, CD54, CD80, CD40, CD58, and MHC class II expression and decreased CD206 expression. The B box caused increased secretion of the proinflammatory cytokines IL-12, IL-6, IL-1alpha, IL-8, TNF-alpha, and RANTES. B box up-regulated CD83 expression as well as IL-6 secretion via a p38 MAPK-dependent pathway. In the MLR, B box-activated DCs acted as potent stimulators of allogeneic T cells, and the magnitude of the response was equivalent to DCs activated by exposure to LPS, nonmethylated CpG oligonucleotides, or CD40L. Furthermore, B box induced secretion of IL-12 from DCs as well as IL-2 and IFN-gamma secretion from allogeneic T cells, suggesting a Th1 bias. HMGB1 released by necrotic cells may be a signal of tissue or cellular injury that, when sensed by DCs, induces and/or enhances an immune reaction.     

Papillomavirus-like particles induce acute activation of dendritic cells

The role of viral structural proteins in the initiation of adaptive immune responses is poorly understood. To address this issue, we focused on the effect of noninfectious papillomavirus-like particles (VLPs) on dendritic cell (DC) activation. We found that murine bone marrow-derived dendritic cells (BMDCs) effectively bound and rapidly internalized bovine papillomavirus VLPS: Exposure to fully assembled VLPs of bovine papillomavirus, human papillomavirus (HPV)16 or HPV18, but not to predominately disordered HPV16 capsomers, induced acute phenotypic maturation of BMDCS: Structurally similar polyomavirus VLPs bound to the DC surface and were internalized, but failed to induce maturation. DCs that had incorporated HPV16 VLPs produced proinflammatory cytokines IL-6 and TNF-alpha; however, the release of these cytokines was delayed relative to LPS activation. Production of IL-12p70 by VLP-exposed DCs required the addition of syngeneic T cells or rIFN-gamma. Finally, BMDCs pulsed with HPV16 VLPs induced Th1-dominated primary T cell responses in vitro. Our data provide evidence that DCs respond to intact papillomavirus capsids and that they play a central role in VLP-induced immunity. These results offer a mechanistic explanation for the striking ability of papillomavirus VLP-based vaccines to induce potent T and B cell responses even in the absence of adjuvant.     

Streptococcal preparation OK-432: a new maturation factor of monocyte-derived dendritic cells for clinical use

For vaccinations based on dendritic cells (DCs), maturation of DCs is critical to the induction of T-cell responses. We tested the efficacy of streptococcal preparation OK-432 as a Good Manufacturing Practice (GMP)-grade maturation agent. OK-432 is currently used in Japan as a cancer immunotherapy drug. Immature monocyte-derived dendritic cells (imMo-DCs) isolated from human peripheral blood monocytes stimulated with granulocyte-macrophage colony stimulating factor and interleukin-4 were exposed to maturation factors, i.e., lipopolysaccharide (LPS), tumor necrosis factor alpha (TNF-alpha) plus prostaglandin E2 (PGE2), and OK-432 for 2 days. OK-432 increased expression of activation- and maturation-related molecules such as HLA-DR, CD80, CD83, and CD86 in imMo-DCs at levels similar to that of TNF-alpha plus PGE2, and higher than that of LPS. All agents examined induced allogeneic T-cell proliferation at a similar level. Only OK-432 caused significant production of interleukin-12 (IL-12) p70 and interferon gamma (IFN-gamma) at both the mRNA and protein levels in imMo-DCs. Neutralizing antibody against IL-12 p70 blocked IFN-gamma secretion from OK-432-stimulated Mo-DCs. IL-12 p70 produced by OK-432-stimulated imMo-DCs induced secretion of IFN-gamma by CD4+ T cells. OK-432 and LPS activated nuclear factor kappa B (NF-kappaB) in imMo-DCs. Both secretion of IL-12 p70 and IFN-gamma and activation of NF-kappaB induced by OK-432 were suppressed when imMo-DCs were pretreated with cytochalasin B. These results indicate that uptake of OK-432 by imMo-DCs is an early critical event for IL-12 p70 production and that NF-kappaB activation induced by OK-432 also contributes partially to IL-12 p70 production. In conclusion, OK-432 is a GMP-grade maturation agent and may be a potential tool for DC-based vaccine therapies.     

Augmentation of antigen-presenting and Th1-promoting functions of dendritic cells by WSX-1(IL-27R) deficiency

WSX-1 is the alpha subunit of the IL-27R complex expressed by T, B, NK/NKT cells, as well as macrophages and dendritic cells (DCs). Although it has been shown that IL-27 has both stimulatory and inhibitory effects on T cells, little is known on the role of IL-27/WSX-1 on DCs. LPS stimulation of splenic DCs in vivo resulted in prolonged CD80/CD86 expression on WSX-1-deficient DCs over wild-type DCs. Upon LPS stimulation in vitro, WSX-1-deficient DCs expressed Th1-promoting molecules higher than wild-type DCs. In an allogeneic MLR assay, WSX-1-deficient DCs were more potent than wild-type DCs in the induction of proliferation of and IFN-gamma production by responder cell proliferation. When cocultured with purified NK cells, WSX-1-deficient DCs induced higher IFN-gamma production and killing activity of NK cells than wild-type DCs. As such, Ag-pulsed WSX-1-deficient DCs induced Th1-biased strong immune responses over wild-type DCs when transferred in vivo. WSX-1-deficient DCs were hyperreactive to LPS stimulation as compared with wild-type DCs by cytokine production. IL-27 suppressed LPS-induced CD80/86 expression and cytokine production by DCs in vitro. Thus, our study demonstrated that IL-27/WSX-1 signaling potently down-regulates APC function and Th1-promoting function of DCs to modulate overall immune responses.