IL-2 is required for the activation of memory CD8+ T cells via antigen cross-presentation

Dendritic cells (DCs) are capable of capturing exogenous Ag for the generation of MHC class I/peptide complexes. For efficient activation of memory CD8(+) T cells to occur via a cross-presentation pathway, DCs must receive helper signals from CD4(+) T cells. Using an in vitro system that reflects physiologic recall memory responses, we have evaluated signals that influence helper-dependent cross-priming, while focusing on the source and cellular target of such effector molecules. Concerning the interaction between CD4(+) T cells and DCs, we tested the hypothesis that CD40 engagement on DCs is critical for IL-12p70 (IL-12) production and subsequent stimulation of IFN-gamma release by CD8(+) T cells. Although CD40 engagement on DCs, or addition of exogenous IL-12 are both sufficient to overcome the lack of help, neither is essential. We next evaluated cytokines and chemokines produced during CD4(+) T cell/DC cross talk and observed high levels of IL-2 produced within the first 18-24 h of Ag-specific T cell engagement. Functional studies using blocking Abs to CD25 completely abrogated IFN-gamma production by the CD8(+) T cells. Although required, addition of exogenous IL-2 did not itself confer signals sufficient to overcome the lack of CD4(+) T cell help. Thus, these data support a combined role for Ag-specific, cognate interactions at the CD4(+) T cell/DC as well as the DC/CD8(+) T cell interface, with the helper effect mediated by soluble noncognate signals.     

Dendritic cells and CD28 costimulation are required to sustain virus-specific CD8+ T cell responses during the effector phase in vivo

Although much is known about the initiation of immune responses, much less is known about what controls the effector phase. CD8(+) T cell responses are believed to be programmed in lymph nodes during priming without any further contribution by dendritic cells (DCs) and Ag. In this study, we report the requirement for DCs, Ag, and CD28 costimulation during the effector phase of the CD8(+) T cell response. Depleting DCs or blocking CD28 after day 6 of primary influenza A virus infection decreases the virus-specific CD8(+) T cell response by inducing apoptosis, and this results in decreased viral clearance. Furthermore, effector CD8(+) T cells adoptively transferred during the effector phase fail to expand without DC, CD28 costimulation, and cognate Ag. The absence of costimulation also leads to reduced survival of virus-specific effector cells as they undergo apoptosis mediated by the proapoptotic molecule Bim. Finally, IL-2 treatment restored the effector response in the absence of CD28 costimulation. Thus, in contrast to naive CD8(+) T cells, which undergo an initial Ag-independent proliferation, effector CD8(+) T cells expanding in the lungs during the effector phase require Ag, CD28 costimulation, and DCs for survival and expansion. These requirements would greatly impair effector responses against viruses and tumors that are known to inhibit DC maturation and in chronic infections and aging where CD28(-/-) CD8(+) T cells accumulate.     

Differential regulation of human blood dendritic cell subsets by IFNs

Based on the relative expression of CD11c and CD1a, we previously identified subsets of dendritic cells (DCs) or DC precursors in human peripheral blood. A CD1a(+)/CD11c(+) population (CD11c(+) DCs), also called myeloid DCs, is an immediate precursor of Langerhans cells, whereas a CD1a(-)/CD11c(-) population (CD11c(-) DCs), sometimes called lymphoid DCs but better known as plasmacytoid DCs, is composed of type I IFN (IFN-alpha beta)-producing cells. Here, we investigate the effects of IFN-alpha beta and IFN-gamma as well as other cytokines on CD11c(+) and CD11c(-) DC subsets, directly isolated from the peripheral blood, instead of in vitro-generated DCs. IFN-gamma and IFN-alpha, rather than GM-CSF, were the most potent cytokines for enhancing the maturation of CD11c(+) DCs. Incubation of CD11c(+) DCs with IFN-gamma also resulted in increased IL-12 production, and this IL-12 allowed DCs to increase Th1 responses by alloreactive T cells. In contrast, IFN-alpha did not induce IL-12 but, rather, augmented IL-10 production. IFN-alpha-primed matured CD11c(+) DCs induced IL-10-producing regulatory T cells; however, this process was independent of the DC-derived IL-10. On the other hand, IFN-alpha by itself neither matured CD11c(-) DCs nor altered the polarization of responding T cells, although this cytokine was a potent survival factor for CD11c(-) DCs. Unlike IFN-alpha, IL-3 was a potent survival factor and induced the maturation of CD11c(-) DCs. The IL-3-primed CD11c(-) DCs activated T cells to produce IL-10, IFN-gamma, and IL-4. Thus, CD11c(+) and CD11c(-) DC subsets play distinct roles in the cytokine network, especially their responses to IFNs.     

Phosphatidylserine regulates the maturation of human dendritic cells

Phosphatidylserine (PS), which is exposed on the surface of apoptotic cells, has been implicated in immune regulation. However, the effects of PS on the maturation and function of dendritic cells (DCs), which play a central role in both immune activation and regulation, have not been described. Large unilamellar liposomes containing PS or phosphatidylcholine were used to model the plasma membrane phospholipid composition of apoptotic and live cells, respectively. PS liposomes inhibited the up-regulation of HLA-ABC, HLA-DR, CD80, CD86, CD40, and CD83, as well as the production of IL-12p70 by human DCs in response to LPS. PS did not affect DC viability directly but predisposed DCs to apoptosis in response to LPS. DCs exposed to PS had diminished capacity to stimulate allogeneic T cell proliferation and to activate IFN-gamma-producing CD4(+) T cells. Exogenous IL-12 restored IFN-gamma production by CD4(+) T cells. Furthermore, activated CTLs proliferated poorly to cognate Ag presented by DCs exposed to PS. Our findings suggest that PS exposure provides a sufficient signal to inhibit DC maturation and to modulate adaptive immune responses.     

The role of the p38 mitogen-activated protein kinase, extracellular signal-regulated kinase, and phosphoinositide-3-OH kinase signal transduction pathways in CD40 ligand-induced dendritic cell activation and expansion of virus-specific CD8+ T cell memory responses

Mature dendritic cells (DCs) are central to the development of optimal T cell immune responses. CD40 ligand (CD40L, CD154) is one of the most potent maturation stimuli for immature DCs. We studied the role of three signaling pathways, p38 mitogen-activated protein kinase (MAPK), extracellular signal-regulated kinase (ERK), and phosphoinositide-3-OH kinase (PI3K), in CD40L-induced monocyte-derived DC activation, survival, and expansion of virus-specific CD8(+) T cell responses. p38 MAPK pathway was critical for CD40L-mediated up-regulation of CD83, a marker of DC maturation. CD40L-induced monocyte-derived DC IL-12 production was mediated by both the p38 MAPK and PI3K pathways. CD40L-mediated DC survival was mostly mediated by the PI3K pathway, with smaller contributions by p38 MAPK and ERK pathways. Finally, the p38 MAPK pathway was most important in mediating CD40L-stimulated DCs to induce strong allogeneic responses as well as expanding virus-specific memory CD8(+) T cell responses. Thus, although the p38 MAPK, PI3K, and ERK pathways independently affect various parameters of DC maturation induced by CD40L, the p38 MAPK pathway within CD40L-conditioned DCs is the most important pathway to maximally elicit T cell immune responses. This pathway should be exploited in vivo to either completely suppress or enhance CD8(+) T cell immune responses.     

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.     

Folate deficiency affects dendritic cell function and subsequent T helper cell differentiation

Insufficient folate status may be related to the increasing prevalence of immune- or inflammation-related chronic diseases. To investigate the effects of folate on immune regulation, we examined the impact of folate deficiency (FD) on dendritic cell (DC) maturation and function and, thus, T helper (Th) cells differentiation. First, bone marrow-derived DCs (BMDCs) were generated from BALB/c mice bone marrow cells cultured in folate-containing (F-BMDCs) or folate-deficient (FD-BMDCs) medium. FD-BMDC displayed more immature phenotype including reduced levels of major histocompatibility complex class II (MHC II), co-stimulatory molecules and characteristic of higher endocytic activity. FD-BMDC produced less IL-12p70 and proinflammatory cytokines in response to lipopolysaccharide. This aberrant DC maturation due to FD resulted in reduced BMDC-induced Th cell activity and lower IL-2, IFNγ, IL-13 and IL-10 productions. Further in vivo study confirmed significantly lower IFNγ and IL-10 productions by T cells and showed higher splenic naïve Th and lower memory T, effector T and regulatory T cell (Treg) percentages in mice fed with the FD diet for 13 weeks. To investigate the role of DCs on T cell activity, splenic DCs (spDC) from FD mice were cocultured with Th cells. The FD spDC had lower MHC II and CD80 expressions and subsequently impaired DC-induced Th differentiation, shown as decreased cytokine productions. This study demonstrated that folate deficiency impaired DC functions and, thus, Th differentiation and responses, suggesting that folate plays a crucial role in maintaining Th cells homeostasis.     

Modulation of CD4 Th cell differentiation by ganglioside GD1a in vitro

Cell surface gangliosides are shed by tumors into their microenvironment. In this study they inhibit cellular immune responses, including APC development and function, which is critical for Th1 and Th2 cell development. Using human dendritic cells (DCs) and naive CD4(+) T cells, we separately evaluated Th1 and Th2 development under the selective differentiating pressures of DC1-inducing pertussis toxin (PT) and DC2-inducing cholera toxin (CT). High DC IL-12 production after PT exposure and high DC IL-10 production after CT exposure were observed, as expected. However, when DCs were first preincubated with highly purified G(D1a) ganglioside, up-regulation of costimulatory molecules was blunted, and PT-induced IL-12 production was reduced, whereas CT-induced IL-10 production was increased. The combination of these effects could contribute to a block in the Th1 response. In fact, when untreated naive T cells were coincubated with ganglioside-preincubated, Ag-exposed DCs, naive Th cell differentiation into Th effector cells was reduced. Both the subsequent DC1-induced T cell production of IFN-gamma (Th1 marker) and DC2-induced T cell IL-4 production (Th2) were inhibited. Thus, ganglioside exposure of DC impairs, by at least two distinct mechanisms, the ability to induce Th differentiation, which could adversely affect the development of an effective cellular antitumor immune response.     

CD40 ligand enhances dengue viral infection of dendritic cells: a possible mechanism for T cell-mediated immunopathology

We have previously shown that dengue virus (DV) productively infects immature human dendritic cells (DCs) through binding to cell surface DC-specific ICAM-3-grabbing nonintegrin molecules. Infected DCs are apoptotic, refractory to TNF-alpha stimulation, inhibited from undergoing maturation, and unable to stimulate T cells. In this study, we show that maturation of infected DCs could be restored by a strong stimulus, CD40L. Addition of CD40L significantly reduced apoptosis of DCs, promoted IL-12 production, and greatly elevated the IFN-gamma response of T cells, but yet did not restore T cell proliferation in MLR. Increased viral infection of DCs was also observed; however, increased infection did not appear to be mediated by DC-specific ICAM-3-grabbing nonintegrin, but rather was regulated by decreased production of IFN-alpha and decreased apoptotic death of infected DCs. Because CD40L is highly expressed on activated memory (but not naive) T cells, the observation that CD40L signaling results in enhanced DV infection of DC suggests a possible T cell-dependent mechanism for the immune-mediated enhancement of disease severity associated with some secondary dengue infections.     

Requirement for the lymphocyte semaphorin,CD100, in the induction of antigen-specific T cells and the maturation of dendritic cells

CD100 belongs to the semaphorin family, several members of which are known to act as repulsive axonal guidance factors during neuronal development. We have previously demonstrated that CD100 plays a crucial role in humoral immunity. In this study, we show that CD100 is also important for cellular immunity through the maturation of dendritic cells (DCs). CD100(-/-) mice fail to develop experimental autoimmune encephalomyelitis induced by myelin oligodendrocyte glycoprotein peptide, because myelin oligodendrocyte glycoprotein-specific T cells are not generated in the absence of CD100. In vitro studies with T cells from OVA-specific TCR-transgenic mice demonstrate that Ag-specific T cells lacking CD100 fail to differentiate into cells producing either IL-4 or IFN-gamma in the presence of APCs and OVA peptide. In addition, DCs from CD100(-/-) mice display poor allostimulatory capabilities and defects in costimulatory molecule expression and IL-12 production. The addition of exogenous soluble rCD100 restores normal functions in CD100(-/-) DCs and further enhances functions of normal DCs. Furthermore, treatment of Ag-pulsed DCs with both soluble CD100 and anti-CD40 before immunization significantly enhances their immunogenicity. This treatment elicits improved T cell priming in vivo, enhancing both primary and memory T cell responses. Collectively, these results demonstrate that CD100, which enhances the maturation of DCs, is essential in the activation and differentiation of Ag-specific T cells.     

The TNF superfamily members LIGHT and CD154 (CD40 ligand) costimulate induction of dendritic cell maturation and elicit specific CTL activity

LIGHT is a recently identified member of the TNF superfamily that is up-regulated upon activation of T cells. Herpesvirus entry mediator, one of its receptors, is constitutively expressed on immature dendritic cells (DCs). In this report, we demonstrate that LIGHT induces partial DC maturation as demonstrated by Ag presentation and up-regulation of adhesion and costimulatory molecules. LIGHT-stimulated DCs show reduced macropinocytosis and enhanced allogeneic stimulatory capacity but fail to produce significant amounts of IL-12, IL-6, IL-1beta, or TNF-alpha compared with unstimulated DCs. However, LIGHT cooperates with CD154 (CD40 ligand) in DC maturation, with particular potentiation of allogeneic T cell proliferation and cytokine secretion of IL-12, IL-6, and TNF-alpha. Moreover, LIGHT costimulation allows DCs to prime in vitro-enhanced specific CTL responses. Our results suggest that LIGHT plays an important role in DC-mediated immune responses by regulating CD154 signals and represents a potential tool for DC-based cancer immunotherapy.     

The Lyn tyrosine kinase differentially regulates dendritic cell generation and maturation

The Src family kinase Lyn plays both stimulatory and inhibitory roles in hemopoietic cells. In this report we provide evidence that Lyn is involved in dendritic cell (DC) generation and maturation. Loss of Lyn promoted DC expansion in vitro from bone marrow precursors due to enhanced generation and accelerated differentiation of Lyn-deficient DC progenitors. Differentiated Lyn-deficient DCs also had a higher survival rate. Similarly, the CD11c-positive cell number was increased in aged Lyn-deficient mice in vivo. In contrast to their enhanced generation, lyn-/- DCs failed to mature appropriately in response to innate stimuli, resulting in DCs with lower levels of MHC class II and costimulatory molecules. In addition, IL-12 production and Ag-specific T cell activation were reduced in lyn-/- DCs after maturation, resulting in impaired Th1 responses. This is the first study to characterize Lyn-deficient DCs. Our results suggest that Lyn kinase plays uniquely negative and positive regulatory roles in DC generation and maturation, respectively.     

An IFN-gamma-IL-18 signaling loop accelerates memory CD8+ T cell proliferation

Rapid proliferation is one of the important features of memory CD8(+) T cells, ensuring rapid clearance of reinfection. Although several cytokines such as IL-15 and IL-7 regulate relatively slow homeostatic proliferation of memory T cells during the maintenance phase, it is unknown how memory T cells can proliferate more quickly than na?ve T cells upon antigen stimulation. To examine antigen-specific CD8(+) T cell proliferation in recall responses in vivo, we targeted a model antigen, ovalbumin(OVA), to DEC-205(+) dendritic cells (DCs) with a CD40 maturation stimulus. This led to the induction of functional memory CD8(+) T cells, which showed rapid proliferation and multiple cytokine production (IFN-gamma, IL-2, TNF-alpha) during the secondary challenge to DC-targeted antigen. Upon antigen-presentation, IL-18, an IFN-gamma-inducing factor, accumulated at the DC:T cell synapse. Surprisingly, IFN-gamma receptors were required to augment IL-18 production from DCs. Mice genetically deficient for IL-18 or IFN-gamma-receptor 1 also showed delayed expansion of memory CD8(+) T cells in vivo. These results indicate that a positive regulatory loop involving IFN-gamma and IL-18 signaling contributes to the accelerated memory CD8(+) T cell proliferation during a recall response to antigen presented by DCs.     

Defective dendritic cell function in HIV-infected patients receiving effective highly active antiretroviral therapy: neutralization of IL-10 production and depletion of CD4+CD25+ T cells restore high levels of HIV-specific CD4+ T cell responses induced by dendritic cells generated in the presence of IFN-alpha

We previously demonstrated that GM-CSF/IFN-alpha combination allowed the differentiation of monocytes from HIV-infected patients into dendritic cells (DCs) exhibiting high CD8(+) T cell stimulating abilities. The present study was aimed at characterizing the ability of DCs generated in the presence of GM-CSF and IFN-alpha to induce CD4 T cell responses. DCs were generated from monocytes of HIV-infected patients in the presence of GM-CSF with either IFN-alpha (IFN-DCs) or IL-4 (IL-4-DCs) for 7 days. Eleven patients receiving highly active antiretroviral therapy and exhibiting CD4 cell counts above 400/mm(3) and plasma HIV-RNA <50 copies/ml for at least 1 year were included in the study. Both DC populations were found to be defective in inducing autologous (in response to tuberculin or HIV-p24) or allogeneic CD4 T cell proliferation. Neutralization of IL-10 during the differentiation of IFN-DCs, but not during the DC-T cell coculture, significantly increased their ability to stimulate autologous CD4 T cell proliferation in response to tuberculin and allogeneic CD4 T cell proliferation (4.1-fold and 3.0-fold increases, respectively, at the DC to T cell ratio of 1:10). Moreover, IL-10 neutralization and CD4(+)CD25(+) T cell depletion synergistically act to dramatically increase HIV-p24-specific CD4 T cell responses induced by IFN-DCs (31.7-fold increase) but not responses induced by IL-4-DCs. Taken together, our results indicate that IFN-DCs are more efficient than IL-4-DCs to stimulate CD4(+) T cell proliferation, further supporting their use for immune-based therapy in HIV infection.     

Modulation of proinflammatory responses to Pneumocystis carinii f. sp. muris in neonatal mice by granulocyte-macrophage colony-stimulating factor and IL-4: role of APCs

Clearance of Pneumocystis carinii f. sp. muris (PC) organisms from the lungs of neonatal mice is delayed due to failure of initiation of inflammation over the first 3 wk after infection. The ability of neonatal lung CD11c(+) dendritic cells (DCs) to induce Ag-specific T cell proliferative responses was significantly reduced compared with adult lung DCs. However, neonatal bone marrow-derived DCs were as competent at presenting PC Ag as were adult bone marrow-derived DCs. Because GM-CSF mRNA expression and activity were significantly reduced in neonatal lungs compared with adults, we treated neonates with exogenous GM-CSF and IL-4 and found enhanced clearance of PC compared with untreated neonates. This was associated with increased lung TNF-alpha, IL-12p35, and IL-18 mRNA expression, indicating enhanced innate immune responses. Cytokine-treated mice had marked expansion of CD11c(+) DCs with up-regulated MHC-II in the lungs. Moreover, increased numbers of activated CD4(+)CD44(high)CD62L(low) cells in the lungs and draining lymph nodes suggested improved Ag presentation by the APCs. Together these data indicate that neonatal lungs lack maturation factors for efficient cellular functioning, including APC maturation.     

Mature dendritic cells infiltrate the T cell-rich region of oral mucosa in chronic periodontitis: in situ, in vivo, and in vitro studies

Previous studies have analyzed the lymphoid and myeloid foci within the gingival mucosa in health and chronic periodontitis (CP); however, the principal APCs responsible for the formation and organizational structure of these foci in CP have not been defined. We show that in human CP tissues, CD1a(+) immature Langerhans cells predominantly infiltrate the gingival epithelium, whereas CD83(+) mature dendritic cells (DCs) specifically infiltrate the CD4(+) lymphoid-rich lamina propria. In vivo evidence shows that exacerbation of CP results in increased levels of proinflammatory cytokines that mediate DC activation/maturation, but also of counterregulatory cytokines that may prevent a Th-polarized response. Consistently, in vitro-generated monocyte-derived DCs pulsed with Porphyromonas gingivalis strain 381 or its LPS undergo maturation, up-regulate accessory molecules, and release proinflammatory (IL-1beta, PGE(2)) and Th (IL-10, IL-12) cytokines. Interestingly, the IL-10:IL-12 ratio elicited from P. gingivalis-pulsed DCs was 3-fold higher than that from Escherichia coli-pulsed DCs. This may account for the significantly (p < 0.05) lower proliferation of autologous CD4(+) T cells and reduced release of IFN-gamma elicited by P. gingivalis-pulsed DCs. Taken together, these findings suggest a previously unreported mechanism for the pathophysiology of CP, involving the activation and in situ maturation of DCs by the oral pathogen P. gingivalis, leading to release of counterregulatory cytokines and the formation of T cell-DC foci.     

Butyrate interferes with the differentiation and function of human monocyte-derived dendritic cells

Dendritic cells (DCs) are specialized antigen-presenting cells that are uniquely capable of either inducing immune responses or maintaining a state of self-tolerance, depending on their stage of maturation. In the present study, we describe a way to interfere with DCs maturation. The compound butyrate can affect the differentiation of DCs generated from human monocytes and can inhibit T cell proliferation. We demonstrate that butyrate substantially down-regulates the expression of CD80, CD83, and MHC class II molecules; increases endocytic capability; reduces allostimulatory abilities; promote interleukin-10 (IL-10) production; and inhibits interleukin-12 (IL-12) and interferon-γ (IFN-γ) production. These results demonstrate a specific immune suppression property of butyrate and supports further investigation for butyrate as a new immunotherapeutic agent.