Cholera toxin induces maturation of human dendritic cells and licences them for Th2 priming

Cholera toxin (CT) is a potent mucosal adjuvant that amplifies B and T cell responses to mucosally co-administered antigens, stimulating predominant Th2-type responses. However, little is known about the mechanism of adjuvanticity of CT and on the influence this toxin may have on Th2 cell development during the priming of an immune response. We analyzed the effect of CT on dendritic cells (DC), which are responsible for the priming of immune responses at the systemic as well as at the mucosal level. We found that CT induces phenotypic and functional maturation of blood monocyte-derived DC. Indeed, CT-treated DC up-regulate expression of HLA-DR molecules, B7. 1 and B7.2 co-stimulatory molecules, and are able to prime naive CD4(+)CD45RA(+) T cells in vitro, driving their polarization towards the Th2 phenotype. Furthermore, CT-matured DC express functional chemokine receptors CCR7 and CXCR4 which may render them responsive to migratory stimuli towards secondary lymphoid organs. Interestingly, the maturation program induced by CT is unique since CT does not induce but rather inhibits cytokine (IL-12p70 and TNF-alpha) and chemokine (RANTES, MIP-1alpha and MIP-1beta) secretion by lipopolysaccharide- or CD40 ligand-activated DC. Our results help to elucidate the mechanism of action of CT as an adjuvant and highlight a new stimulus of bacterial origin that promotes maturation of DC.     

Frontline: Peripheral priming of alloreactive T cells by the direct pathway of allorecognition

Recent studies, though controversial, have suggested that secondary lymphoid organs may not constitute an essential site for the initiation of immune responses to transplant antigens. However, this issue has never been examined in the context of direct and indirect allorecognition. Here, we characterized immune responses arising in draining lymph nodes and skin allografts, in a murine model based on a single T cell clonotype where these two pathways can be independently studied. In this model, graft rejection by the direct or the indirect pathway occurred with similar kinetics, although initiation of the alloreactive responses was clearly different. During indirect responses, expansion and activation of alloreactive T cells were first observed in draining lymph nodes, at day 7 post-transplant, and graft-infiltrating T cells were observed later, at day 11. In striking contrast, directly activated alloreactive T cells were detected at an early stage inside the graft, and only later in the draining lymph nodes, after skin allograft rejection was almost completed. These results suggest that sensitization of naive T cells through the direct pathway could take place outside secondary lymphoid organs.     

Th1 or Th2 balance regulated by interaction between dendritic cells and NKT cells

If Th1 or Th2 polarization could be artificially manipulated, effective immune responses would be generated depending on nature of the targets. In this study we attempted to regulate CD40 expressions on dendritic cells (DCs) in order to modify the T cell response. It was found that reducing agents selectively inhibited surface expression of CD40 on DCs. This finding may provide a new strategy of DC-mediated modulation of the Th1/Th2 balance. It was also shown that NKT-produced Th1/Th2 cytokine balance was under control of negative feedback loop through DCs. Th1 cytokine-pretreated DCs mainly induced Th2 cytokine production, whereas Th2 cytokine-pretreated DCs induced Th1 cytokine production by α-galactosylceramide-stimulated NKT cells. The negative feedback regulation system could be applicable to therapeutics of various diseases based on immunological disorders. Presented at the First Robert A Good Society Symposium, St. Petersburg, FL 2006.     

直接和间接共培养条件下小鼠子宫自然杀伤细胞与树突状细胞相互作用的比较

背景：子宫自然杀伤细胞与树突状细胞可以相互作用,但相互间作用是否必须直接接触还存在不同的观点。 目的：对比直接和间接共培养条件下子宫自然杀伤细胞与树突状细胞之间的相互作用,明确两者相互作用的方式。 方法：将树突状细胞和子宫自然杀伤细胞进行直接接触共培养,设为直接共培养组;在2种细胞Transwell系统中进行共培养,设为间接共培养组。观察培养细胞的生长状况,酶联吸附法检测培养上清白细胞介素10,12、转化生长因子β细胞因子的浓度,流式细胞术检测各组细胞表面标志CD86的表达情况。 结果与结论：与单独培养树突状细胞和子宫自然杀伤细胞相比,直接和间接共培养组培养液上清中白细胞介素10,12、转化生长因子β浓度均增加(P < 0.05),尤以白细胞介素10浓度增加明显(P < 0.05),CD86的表达明显增高 (P < 0.05)。直接共培养组白细胞介素10,12和转化生长因子β浓度更高(P < 0.05),表达CD86的细胞含量更多 (P < 0.05)。证实子宫自然杀伤细胞可以促进树突状细胞成熟,而树突状细胞促进子宫自然杀伤细胞的分泌,两者通过细胞间的直接接触而相互作用。     

Coordinate NF-kappaB and STAT1 activation promotes development of myeloid type 1 dendritic cells

NF-κB and STAT1 are critically involved in the initiation of the inflammatory cascade. Using semi-automated imaging cytometry and fluorescent antibodies, we screened several factors for their ability to induce nuclear translocation of RelA/NF-κB and STAT1 in subsets of monocyte-derived dendritic cells (DC). Detailed kinetics and dose–response studies are presented for IL-1-, LPS-, CD40L-, IFN-γ- and IFN-α-stimulated responses. The results are consistent with the notion that simultaneous activation of both STAT1 and NF-κB pathways at the initiation of differentiation culture is required for efficient priming of IL-12 production by DC. Maturation of DC led to characteristic NF-κB and STAT1 distribution and response patterns. During the resting stage, DC differentiated under the presence of IFN-γ showed sustained STAT activation and remained responsive to LPS. By contrast, PGE₂-supplemented DC could be characterized by negligible responses to LPS and IFN-γ and a remarkable NF-κB response to CD40L. STAT1 pathway was suppressed in PGE₂-supplemented cells. We conclude that the magnitude and temporal kinetics of the nuclear shift of STAT1 and NF-κB in myeloid DC are associated with IL-12p70 production and are dependent on the nature of the stimulating factors and the polarization state of cells.     

4-1BB costimulation promotes bystander activation of human CD8 T cells

Costimulatory signals potently promote T-cell proliferation and effector function. Agonistic antibodies targeting costimulatory receptors of the TNFR family, such as 4-1BB and CD27, have entered clinical trials in cancer patients. Currently there is limited information how costimulatory signals regulate antigen-specific but also bystander activation of human CD8 T cells. Engineered antigen presenting cells (eAPC) efficiently presenting several common viral epitopes on HLA-A2 in combination with MHC class I tetramer staining were used to investigate the impact of costimulatory signals on human CD8 T-cell responses. CD28 costimulation potently augmented the percentage and number of antigen-reactive CD8 T cells, whereas eAPC expressing 4-1BB-ligand induced bystander proliferation of CD8 T cells and massive expansion of NK cells. Moreover, the 4-1BB agonist urelumab similarly induced bystander proliferation of CD8 T cells and NK cells in a dose-dependent manner. However, the promotion of bystander CD8 T-cell responses is not a general attribute of costimulatory TNF receptor superfamily (TNFRSF) members, since CD27 signals enhanced antigen-specific CD8 T cells responses without promoting significant bystander activation. Thus, the differential effects of costimulatory signals on the activation of human bystander CD8 T cells should be taken into account when costimulatory pathways are harnessed for cancer immunotherapy.     

Soluble flagellin coimmunization attenuates Th1 priming to Salmonella and clearance by modulating dendritic cell activation and cytokine production

Soluble flagellin (sFliC) from Salmonella Typhimurium (STm) can induce a Th2 response to itself and coadministered antigens through ligation of TLR5. These properties suggest that sFliC could potentially modulate responses to Th1 antigens like live STm if both antigens are given concurrently. After coimmunization of mice with sFliC and STm there was a reduction in Th1 T cells (T‐bet⁺IFN‐γ⁺ CD4 T cells) compared to STm alone and there was impaired clearance of STm. In contrast, there was no significant defect in the early extrafollicular B‐cell response to STm. These effects are dependent upon TLR5 and flagellin expression by STm. The mechanism for these effects is not related to IL‐4 induced to sFliC but rather to the effects of sFliC coimmunization on DCs. After coimmunization with STm and sFliC, splenic DCs had a lower expression of costimulatory molecules and profoundly altered kinetics of IL‐12 and TNFα expression. Ex vivo experiments using in vivo conditioned DCs confirmed the effects of sFliC were due to altered DC function during a critical window in the coordinated interplay between DCs and naïve T cells. This has marked implications for understanding how limits in Th1 priming can be achieved during infection‐induced, Th1‐mediated inflammation.     

Effect of multiple activation stimuli on the generation of Th1-polarizing dendritic cells

It was originally reported that only a small fraction of total matured dendritic cells (DCs) produced interleukin (IL)-12, but it has never been determined whether different combinations of activating signals now shown to maximize secreted IL-12 do so through increasing output by the same IL-12 producers, or by recruiting additional cytokine-secreting cells. We therefore tested all combinations of bacterial lipopolysaccharide (LPS) (TLR4 ligand), R848 (TLR8 ligand), interferon (IFN)-γ, and CD40L for activating human monocyte-derived dendritic cells (DC), and determined by intracellular flow cytometry that enhanced IL-12 secretion was accomplished in large part by markedly increasing the proportion of cells producing IL-12, with the triple and quadruple combinations recruiting the most DC. This optimization requirement for multiple signals was not reflected in differential Toll-like receptor (TLR) expression by the cells. Interestingly, DCs activated with single TLR ligands plus IFN-γ were capable of responding with a second burst of IL-12 upon later CD40L stimulation, whereas DCs activated with R848 plus LPS were not, despite the trend of the latter for superior polarization of naive T cells toward IFN-γ-secreting Th1. These results have implications for the biology of IL-12-secreting DCs and choice of activation regimen for prospective use in DC-based immunotherapy.     

Polymorphonuclear neutrophils deliver activation signals and antigenic molecules to dendritic cells: a new link between leukocytes upstream of T lymphocytes

Polymorphonuclear neutrophils (PMNs) are rapidly recruited to tissues upon injury or infection. There, they can encounter local and/or recruited immature dendritic cells (iDCs), a colocalization that could promote at least transient interactions and mutually influence the two leukocyte populations. Using human live blood PMNs and monocyte-derived iDCs, we examined if these leukocytes actually interacted and whether this influenced DC function. Indeed, coculture with live but not apoptotic PMNs led to up-regulation of membrane CD40, CD86, and human leukocyte antigen (HLA)-DR on DCs. Whereas CD40 up-regulation was dependent on soluble factors released by PMNs, as determined in cultures conducted in different chambers, cell contact was necessary for CD86 and HLA-DR up-regulation, a process that was inhibited by anti-CD18 antibodies, indicating that CD18 ligation was required. We also found that via a cell contact-dependent mechanism, DCs acquired Candida albicans-derived antigens from live as well as from apoptotic PMNs and could thus elicit antigen-specific T lymphocyte responses. Altogether, our data demonstrate the occurrence of cross-talk between human PMNs and DCs and provide new insights into the immune processes occurring upstream of the interactions between DCs and T lymphocytes.     

Kinetics of dendritic cell activation: impact on priming of TH1, TH2 and nonpolarized T cells

To prime immune responses, dendritic cells (DCs) need to be activated to acquire T cell stimulatory capacity. Although some stimuli trigger interleukin 12 (IL-12) production that leads to T helper cell type I (TH1) polarization, others fail to do so and favor TH2 polarization. We show that after activation by lipopolysaccharide, DCs produced IL-12 only transiently and became refractory to further stimulation. The exhaustion of cytokine production impacted the T cell polarizing process. Soon after stimulation DCs primed strong TH1 responses, whereas at later time points the same cells preferentially primed TH2 and nonpolarized T cells. These findings indicate that during an immune response, T cell priming conditions may change in the lymph nodes, suggesting another mechanism for the regulation of effector and memory T cells.     

T cell priming by dendritic cells: thresholds for proliferation,differentiation and death and intraclonal functional diversification

The variables that influence priming of human naive CD4+ T cells by dendritic cells (DC) were dissected in vitro by analyzing the response to the bacterial superantigen toxicshock syndrome toxin or to alloantigens. We show that under conditions that force DC-T cell interactions a single DC can prime up to 20 naive T cells. Moreover, the strength of antigenic stimulation, as determined by DC numbers, antigen dose, TCR avidity and duration of DC-T cell interactions, drives the progressive differentiation of proliferating T cells from a non-effector CCR7+ stage, to an effector CCR7- stage and, eventually, to cell death. We also show that the proliferating CCR7+ and CCR7- populations share clonotypic sequences, demonstrating that the two cell fates can be generated within a single clone. Taken together these results indicate that the strength of antigenic stimulation regulates T cell progression through thresholds of proliferation, differentiation and death. However, the random nature of DC-T cell encounters introduces a critical stochastic element in T cell stimulation, which leads to the generation of cells endowed with distinct homing potentials and effector functions within a given T cell clone.     

MV140, a sublingual polyvalent bacterial preparation to treat recurrent urinary tract infections,licenses human dendritic cells for generating Th1, Th17, and IL-10 responses via Syk and MyD88

Recurrent urinary tract infections (RUTIs) are one of the most common bacterial infectious diseases, especially in women. Antibiotics remain the mainstay of treatment, but their overuse is associated with antibiotic-resistant infections and deleterious effects in the microbiota. Therefore, alternative approaches are fully demanded. Sublingual immunization with MV140 (Uromune), a polyvalent bacterial preparation (PBP) of whole heat-inactivated bacteria, demonstrated clinical efficacy for the treatment of RUTIs, but the involved immunological mechanisms remain unknown. Herein, we demonstrated that MV140 endorses human dendritic cells (DCs) with the capacity to generate Th1/Th17 and IL-10-producing T cells by mechanisms depending on spleen tyrosine kinase (Syk)- and myeloid differentiation primary response gene 88 (MyD88)-mediated pathways. MV140-induced activation of nuclear factor κB (NF-κB) and p38 in human DCs is essential for the generated Th1/Th17 and IL-10 immune responses whereas c-Jun N-terminal Kinase (JNK) and extracellular-signal regulated kinase (ERK) contribute to Th1 and IL-10 responses, respectively. Sublingual immunization of BALB/c mice with MV140 also induces potent systemic Th1/Th17 and IL-10 responses in vivo. We uncover immunological mechanisms underlying the way of action of MV140, which might well also contribute to understand the rational use of specific PBPs in other clinical conditions with potential high risk of recurrent infections.     

Relative efficiency of microglia, astrocytes, dendritic cells and B cells in naive CD4+ T cell priming and Th1/Th2 cell restimulation.

We have compared the efficiency of central nervous system and peripheral antigen-presenting cells (APC) in T cell priming and restimulation. OVA peptide 323 - 339-dependent activation of DO11.10 TCR-transgenic naive CD4+ and polarized Th1 or Th2 cells was assessed in the presence of microglia and astrocytes from the neonatal mouse brain as well as dendritic cells (DC) and B cells purified from adult mouse lymph nodes. DC were the most efficient in inducing naive T cell proliferation, IL-2 secretion and differentiation into Th1 cells, followed by IFN-gamma-preactivated microglia, large and small B cells. Astrocytes failed to activate naive T cells. IFN-gamma-pretreated microglia were as efficient as DC in the restimulation of Th1 cells, whereas IFN-gamma-pretreated astrocytes, large and small B cells were much less efficient. Conversely, Th2 cells were efficiently restimulated by all the APC types examined. During T cell priming, DC secreted more IL-12 than microglia but similar amounts of IL-12 were secreted by the two cell types upon interaction with Th1 cells. The hierarchy of APC established in this study indicates that DC and microglia are the most efficient in the stimulation of naive CD4(+) T cells and in the restimulation of Th1 cells, suggesting that activated microglia may effectively contribute to Th1 responses leading to central nervous system inflammation and tissue damage. These potentially pathogenic responses could be counteracted by the high efficiency of astrocytes as well as microglia in restimulating Th2 cells.     

Direct and indirect activation of nerve cells by electrical pulses applied extracellularly.

1. The mode of activation of nerve cells by extracellular stimuli was investigated while recording from a selected cell with one electrode, and applying current pulses around this cell with another electrode. The analysis was done on motoneurones and on spinal border cells from lower lumbar segments in the cat. 2. Directly evoked action potentials were defined by their appearance in an all-or-none fashion with stable latencies of less than 0-5 ms. The lowest thresholds for their generation were 0-15-0-20 muA in the spinal border cells and 0-35-0-40 muA in the motoneurones. In the main series on motoneurones a correlation has been established between different positions of the extracellular stimulating electrode in relation to the cells and the thresholds for the direct excitation of these cells. The position of the electrode were defined on the basis of an analysis of the IS and SD components of the action potentials recorded extracellularly around the cell when evoked by current pulses applied through the intracellular electrode; both the amplitudes of these IS and SD components and their timing with the IS and SD spikes, which were simultaneously recorded with the intracellular electrode, were then taken into account. The lowest thresholds (less than 2 muA) for the direct activation of cells were found nearest the initial segment of the axon. Their values increased to about 5 mu A at near-soma positions and to greater than 10 muA at near-dendrites positions about 150 mum away. 3. Transsynaptically evoked action potentials which were clearly set up by the preceding e.p.s.p.s appeared with latencies greater than 0-7 ms. When single current pulses were used, the lowest thresholds for transsynaptic spike activation were usually greater than 5-10 muA but they considerably decreased with repetitive stimuli. These thresholds were higher than the thresholds for the direct activation of cells within the region of the initial segment, of the same order of magnitude near the soma, and lower when the stimulating electrode was nearer the dendrites than the soma and generally at all larger distances from the cells. 4. All the observations on direct excitation of cells by extracellular stimuli (generation of the IS spike before the SD spike, lowest thresholds near the region of the initial segment of the axon, similar rates of increase in these thresholds with distance as for fibres) lead to the conclusion that the effects of the extracellular stimuli are exerted primarily via spread of current to the initial segment of the axon and its depolarization. 5. Late extracellular negativities presumably related to dendritic activation were observed in a few cells. These negativities were synchronous with late components of the intracellulary recorded action potentials.     

CD4+CD25+Foxp3+ indirect alloreactive T cells from renal transplant patients suppress both the direct and indirect pathways of allorecognition

Alloreactive T cells recognize donor antigens by two routes: direct and indirect pathways of allorecognition. Although the direct pathway is reported to be dominant in allograft rejection, indirect allorecognition also plays an important role. Indirect alloreactivity is also observed in renal transplant patients irrespective of rejection. Previously we showed a predominance of interleukin (IL)-10 induced by indirect allorecognition of donor human leucocyte antigen (HLA)-DR peptides, suggesting the existence of indirect alloreactive T cells displaying regulatory activity. In the present work, our objective was to characterize these regulatory T cells. We detected indirect alloproliferation of peripheral blood mononuclear cells (PBMC) from renal transplant patients, induced by donor HLA-DR peptides, dependent on IL-4 or IL-10, suggesting regulatory activity as part of the alloreactive T-cell repertoire. PBMC-derived indirect alloreactive T-cell lines were established and produced both inflammatory and regulatory cytokines. We showed that two of these T-cell lines which were able to inhibit both direct and indirect alloproliferation of another T-cell line from the same patient presented a CD4(+)CD25(+)Foxp3(+) T-cell population. These data support the idea that indirect alloreactive T cells may also have regulatory activity and may contribute to the maintenance of the human renal allograft.     

Human type 1 and type 2 conventional dendritic cells express indoleamine 2,3-dioxygenase 1 with functional effects on T cell priming

Dendritic cells (DCs) are key regulators of the immune system that shape T cell responses. Regulation of T cell induction by DCs may occur via the intracellular enzyme indoleamine 2,3-dioxygenase 1 (IDO), which catalyzes conversion of the essential amino acid tryptophan into kynurenine. Here, we examined the role of IDO in human peripheral blood plasmacytoid DCs (pDCs), and type 1 and type 2 conventional DCs (cDC1s and cDC2s). Our data demonstrate that under homeostatic conditions, IDO is selectively expressed by cDC1s. IFN-γ or TLR ligation further increases IDO expression in cDC1s and induces modest expression of the enzyme in cDC2s, but not pDCs. IDO expressed by conventional DCs is functionally active as measured by kynurenine production. Furthermore, IDO activity in TLR-stimulated cDC1s and cDC2s inhibits T cell proliferation in settings were DC-T cell cell-cell contact does not play a role. Selective inhibition of IDO1 with epacadostat, an inhibitor currently tested in clinical trials, rescued T cell proliferation without affecting DC maturation status or their ability to cross-present soluble antigen. Our findings provide new insights into the functional specialization of human blood DC subsets and suggest a possible synergistic enhancement of therapeutic efficacy by combining DC-based cancer vaccines with IDO inhibition.