Mast cells promote Th1 and Th17 responses by modulating dendritic cell maturation and function

Mast cells (MCs) play an important role in the regulation of protective adaptive immune responses against pathogens. However, it is still unclear whether MCs promote such host defense responses via direct effects on T cells or rather by modifying the functions of antigen-presenting cells. To identify the underlying mechanisms of the immunoregulatory capacity of MCs, we investigated the impact of MCs on dendritic cell (DC) maturation and function. We found that murine peritoneal MCs underwent direct crosstalk with immature DCs that induced DC maturation as evidenced by enhanced expression of costimulatory molecules. Furthermore, the MC/DC interaction resulted in the release of the T-cell modulating cytokines IFN-γ, IL-2, IL-6 and TGF-β into coculture supernatants and increased the IL-12p70, IFN-γ, IL-6 and TGF-β secretion of LPS-matured DCs. Such MC-"primed" DCs subsequently induced efficient CD4+ T-cell proliferation. Surprisingly, we observed that MC-primed DCs stimulated CD4+ T cells to release high levels of IFN-γ and IL-17, demonstrating that MCs promote Th1 and Th17 responses. Confirming our in vitro findings, we found that the enhanced disease progression of MC-deficient mice in Leishmania major infection is correlated with impaired induction of both Th1 and Th17 cells.     

Optimal culture conditions for the generation of natural killer cell-induced dendritic cells for cancer immunotherapy

Dendritic cell (DC)-based vaccines continue to be considered an attractive tool for cancer immunotherapy. DCs require an additional signal from the environment or other immune cells to polarize the development of immune responses toward T helper 1 (Th1) or Th2 responses. DCs play a role in natural killer (NK) cell activation, and NK cells are also able to activate and induce the maturation of DCs. We investigated the types of NK cells that can induce the maturation and enhanced function of DCs and the conditions under which these interactions occur. DCs that were activated by resting NK cells in the presence of inflammatory cytokines exhibited increased expression of several costimulatory molecules and an enhanced ability to produce IL-12p70. NK cell-stimulated DCs potently induced Th1 polarization and exhibited the ability to generate tumor antigen-specific cytotoxic T lymphocyte responses. Our data demonstrate that functional DCs can be generated by coculturing immature DCs with freshly isolated resting NK cells in the presence of Toll-like receptor agonists and proinflammatory cytokines and that the resulting DCs effectively present antigens to induce tumor-specific T-cell responses, which suggests that these cells may be useful for cancer immunotherapy.     

Dexamethasone turns tumor antigen-presenting cells into tolerogenic dendritic cells with T cell inhibitory functions

BackgroundDendritic cells (DCs) are usually immunogenic, but they are also capable of inducing tolerance under anti-inflammatory conditions. Immunotherapy based on autologous DCs loaded with an allogeneic melanoma cell lysate (TRIMEL/DCs) induces immunological responses and increases melanoma patient survival. Glucocorticoids can suppress DC maturation and function, leading to a DC-mediated inhibition of T cell responses.MethodsThe effect of dexamethasone, a glucocorticoid extensively used in cancer therapies, on TRIMEL/DCs phenotype and immunogenicity was examined.ResultsDexamethasone induced a semi-mature phenotype on TRIMEL/DC with low maturation surface marker expressions, decreased pro-inflammatory cytokine induction (IL-1β and IL-12) and increased release of regulatory cytokines (IL-10 and TGF-β). Dexamethasone-treated TRIMEL/DCs inhibited allogeneic CD4⁺ T cell proliferation and cytokine release (IFNγ, TNF-α and IL-17). Co-culturing melanoma-specific memory tumor-infiltrating lymphocytes with dexamethasone-treated TRIMEL/DC inhibited proliferation and effector T cell activities, including cytokine secretion and anti-melanoma cytotoxicity.ConclusionsThese findings suggest that dexamethasone repressed melanoma cell lysate-mediated DC maturation, generating a potent tolerogenic-like DC phenotype that inhibited melanoma-specific effector T cell activities. These results suggest that dexamethasone-induced immunosuppression may interfere with the clinical efficacy of DC-based melanoma vaccines, and must be taken into account for optimal design of cellular therapy against cancer.     

Leptin deficiency impairs maturation of dendritic cells and enhances induction of regulatory T and Th17 cells

Leptin is an adipose‐secreted hormone that plays an important role in both metabolism and immunity. Leptin has been shown to induce Th1‐cell polarization and inhibit Th2‐cell responses. Additionally, leptin induces Th17‐cell responses, inhibits regulatory T (Treg) cells and modulates autoimmune diseases. Here, we investigated whether leptin mediates its activity on T cells by influencing dendritic cells (DCs) to promote Th17 and Treg‐cell immune responses in mice. We observed that leptin deficiency (i) reduced the expression of DC maturation markers, (ii) decreased DC production of IL‐12, TNF‐α, and IL‐6, (iii) increased DC production of TGF‐β, and (iv) limited the capacity of DCs to induce syngeneic CD4⁺ T‐cell proliferation. As a consequence of this unique phenotype, DCs generated under leptin‐free conditions induced Treg or T_H17 cells more efficiently than DCs generated in the presence of leptin. These data indicate important roles for leptin in DC homeostasis and the initiation and maintenance of inflammatory and regulatory immune responses by DCs.     

Purification of splenic dendritic cells induces maturation and capacity to stimulate Th1 response in vivo

Dendritic cell (DC) maturation state is a key parameter for the issue of DC-T cell cognate interaction, which determines the outcome of T cell activation. Indeed, immature DCs induce tolerance while fully mature DCs generate immunity. Here we show that, in the absence of any deliberate activation signal, DCs freshly isolated from mouse spleen spontaneously produce IL-12 and tumor necrosis factor-alpha and up-regulate co-stimulation molecules, even when directly re-injected into their natural environment. Furthermore, after their isolation, these cells acquire the capacity to induce specific T(h)1 responses in vivo. These results demonstrate that the sole isolation of spleen DCs leads to the full maturation of these cells, which therefore cannot be considered as immature DCs. Moreover, we also show that the kinetics of DC activation do not influence the polarization of T(h) response in vivo challenging the idea that exhausted DCs induce preferentially T(h)2 response. Altogether, these observations should be taken into account in all experiments based on the transfer of ex vivo purified DCs.     

Prolonged exposure of dendritic cells to maturation stimuli favors the induction of type-2 cytotoxic T lymphocytes

Dendritic cell (DC) maturation influences the priming and polarization of T lymphocytes. We recently found that early activated DC (i.e. DC exposed to pro-maturation stimuli for 8 h) were more prone to prime in vivo a type-1 cytotoxic T cell (Tc1) response than DC exposed to pro-maturation stimuli for 48 h (48h-DC). We investigated whether 48h-DC, conversely, allowed the induction of Tc2 cells. Antigen-pulsed mouse bone-marrow-derived DC at any maturation stage, in the presence of exogenous IL-12, skewed in vitro naive CD8(+) T cells towards Tc1 cells, but 48h-DC most potently, in the presence of exogenous IL-4, favored the induction of Tc2 cells. In vivo, full maturation of DC promoted expansion of Tc2 and fall of Tc1 cells. Tc2 cells maintained a high cytolytic activity and produced significant amounts of IL-4, IL-5, IL-10 and TGF-beta. Our results indicate that polarization of naive CD8(+) T cells to Tc2 cells is dependent on the amount of time DC have been exposed to maturation stimuli, and might be favored in late and/or chronic phases of an immune response.     

Helper role of NK cells during the induction of anticancer responses by dendritic cells

Recent reports demonstrate that natural killer (NK) cells and dendritic cells (DC) support each other's activity in a positive feedback. We observed that activated NK cells induce the maturation of DCs into stable type-1 polarized DCs (DC1), characterized by up to 100-fold enhanced ability to produce IL-12p70 in response to subsequent interaction with Th cells. DC1 induction depends on NK cell-produced IFN-gamma and TNF-alpha, with a possible involvement of additional factors. DC1, induced by NK cells or by NK cell-related soluble factors, are stable, resistant to tumor-related suppressive factors, and show strongly enhanced ability to induce Th1 and CTL responses. In analogy to resting T cells, the induction of "helper" function of NK cells relies on a two-signal activation paradigm. While NKG2D-dependent tumor cell recognition is sufficient to induce the cytotoxic "effector" function of NK cells, the induction of "NK cell help" requires additional signals from type-1 IFNs, products of virally-infected cells, or from IL-2. Compared to non-polarized DCs currently-used in clinical trials, DC1s act as superior inducers of anti-cancer CTL responses during in vitro sensitization. The current data provides rationale for the clinical use of DC1s in cancer and chronic infections (such as HIV), as a new generation DC-based vaccines, uniquely combining fully mature DC status with an elevated, rather than "exhausted" ability to produce bioactive IL-12p70. We are currently implementing stage I/II clinical trials, testing the effectiveness of DC1s induced by NK cells or by NK cell-related factors, as therapeutic vaccines against melanoma.     

Natural killer cell and hepatic cell interaction via NKG2A leads to dendritic cell-mediated induction of CD4 CD25 T cells with PD-1-dependent regulatory activities

Natural killer (NK) cells have the ability to control dendritic cell (DC)-mediated T cell responses. However, the precise mechanisms by which NK receptor-mediated regulation of NK cells determines the magnitude and direction of DC-mediated T cell responses remain unclear. In the present study, we applied an in vitro co-culture system to examine the impact of NK cells cultured with hepatic cells on DC induction of regulatory T cells. We found that interaction of NK cells and non-transformed hepatocytes (which express HLA-E) via the NKG2A inhibitory receptor resulted in priming of DCs to induce CD4(+) CD25(+) T cells with regulatory properties. NKG2A triggering led to characteristic changes of the cytokine milieu of co-cultured cells; an increase in the transforming growth factor (TGF)-beta involved in the generation of this specific type of DC, and a decrease in the tumour necrosis factor-alpha capable of antagonizing the effect of TGF-beta. The regulatory cells induced by NK cell-primed DCs exert their suppressive actions through a negative costimulator programmed death-1 (PD-1) mediated pathway, which differs from freshly isolated CD4(+) CD25(+) T cells. These findings provide new insight into the role of NK receptor signals in the DC-mediated induction of regulatory T cells.     

Lysophosphatidic acid modulates the activation of human monocyte-derived dendritic cells

Lysophosphatidic acid (LPA) is a biologically active lysophospholipid that can regulate immune activation. LPA can activate T cells and dendritic cells (DCs), but the effects of LPA on the ability of DCs to influence T cell polarization are not well understood. Human monocyte-derived DCs were differentiated in vitro in the presence of interleukin-4 (IL-4) and granulocyte-macrophage colonystimulating factor (GM-CSF), and matured with LPA and lipopolysaccharide (LPS) alone or in combination. DC activation was monitored by analyzing cell-surface expression of co-stimulatory receptors and cytokine production. The ability of DCs to influence T cell activation was determined using two models of DC:T cell co-culture. Maturation with LPS induced dose-dependent DC activation characterized by enhanced expression of co-stimulatory molecules (e.g., CD86) and production of cytokines including IL-6 and IL-10. Co-incubation with LPA attenuated the LPS-induced production of IL-6, without significantly affecting IL-10 secretion or the ability of DC to promote T cell proliferation. DCs matured in the presence of both LPA and LPS enhanced the production of interferon-gamma (IFN-gamma) when co-cultured with allogeneic T cells, compared with DC matured by LPS alone. Similar results were found using a model of allogeneic na?ve T cell differentiation, where LPA- plus LPS-matured DC enhanced IFN-gamma as well as IL-4 secretion after restimulation. Lysophosphatidic acid fine-tunes the effects of LPS on human myeloid DC maturation, but does not exert a dominant effect on the ability of DC to influence Th cell polarization.     

European genotype of porcine reproductive and respiratory syndrome (PRRSV) infects monocyte-derived dendritic cells but does not induce Treg cells

The aim of this study was to characterize the immune responses of DCs after infection with four different EU strains of PRRSV and whether they show any ability to immunomodulate T cells activation. Our results show that all EU strains can efficiently infect and replicate in DCs. Nevertheless, SLA-II levels remained unaltered in DC infected by all EU PRRSV strains, whereas SLA-I expression was only reduced when strain 2992 was used. IL-10 production was induced by three EU PRRSV strains, being strain 2992 the highest inducer. However, no induction of Treg cells, measured by CD25 and Foxp3 expression on lymphocytes co-cultured with infected DCs, was found. TGF-β induction was not detected in DC infected with any EU strain tested. In conclusion, DCs infected with EU PRRSV strains exhibited an unbalanced ability to stimulate T cell response and was strain dependent. However, Treg cells were not induced, at least in vitro.     

CCL19 and CCL21 induce a potent proinflammatory differentiation program in licensed dendritic cells

Dendritic cells (DCs) are key instigators of adaptive immune responses. Using an alphaviral expression cloning technology, we have identified the chemokine CCL19 as a potent inducer of T cell proliferation in a DC-T cell coculture system. Subsequent studies showed that CCL19 enhanced T cell proliferation by inducing maturation of DCs, resulting in upregulation of costimulatory molecules and the production of proinflammatory cytokines. Moreover, CCL19 programmed DCs for the induction of T helper type (Th) 1 rather than Th2 responses. Importantly, only activated DCs that migrated from the periphery to draining lymph nodes, but not resting steady-state DCs residing within lymph nodes, expressed high levels of CCR7 in vivo and responded to CCL19 with the production of proinflammatory cytokines. Migrating DCs isolated from mice genetically deficient in CCL19 and CCL21 (plt/plt) presented an only partially mature phenotype, highlighting the importance of these chemokines for full DC maturation in vivo. Our findings indicate that CCL19 and CCL21 are potent natural adjuvants for terminal activation of DCs and suggest that chemokines not only orchestrate DC migration but also regulate their immunogenic potential for the induction of T cell responses.     

Rv0315, a novel immunostimulatory antigen of Mycobacterium tuberculosis, activates dendritic cells and drives Th1 immune responses

Tuberculosis (TB) caused by Mycobacterium tuberculosis (Mtb) is one of the most deadly infectious diseases, with approximately two million people dying of TB annually. An effective therapeutic method for activating dendritic cells (DCs) and driving Th1 immune responses would improve host defenses and further the development of a TB vaccine. Given the importance of DC maturation in eliciting protective immunity against TB, we investigated whether Rv0315, a newly identified Mtb antigen, can prompt DC maturation. We found that Rv0315 functionally activated DCs by augmenting the expression of the co-stimulatory molecules CD80 and CD86 as well as MHC class I/II molecules. Moreover, it increased DC secretion of the pro-inflammatory cytokines IL-6, IL-1β, and TNF-α. Unlike LPS, however, Rv0315 induced the secretion of IL-12p70, but not IL-10. In addition, Rv0315-treated DCs accelerated the proliferation of CD4(+) and CD8(+) splenic T cells from Mtb-infected mice, with increased levels of IFN-γ, in syngeneic and allogeneic mixed lymphocyte reactions, indicating that Rv0315 contributes to Th1 polarization of the immune response. Importantly, both mitogen-activated protein kinases and nuclear factor κB signaling mediated the expression of DC surface markers and cytokines. Taken together, our results indicate that Rv0315 is a novel DC maturation-inducing antigen that drives T cell immune responses toward Th1 polarization, suggesting that Rv0315 plays a key role in determining the nature of the immune response to TB.     

Inflammatory signals in dendritic cell activation and the induction of adaptive immunity

Summary:  Pathogen invasion induces a rapid inflammatory response initiated through the recognition of pathogen-derived molecules by pattern recognition receptors (PRRs) expressed on both immune and non-immune cells. The initial wave of pro-inflammatory cytokines and chemokines limits pathogen spread and recruits and activates immune cells to eradicate the invaders. Dendritic cells (DCs) are responsible for initiating a subsequent phase of immunity, dominated by the action of pathogen-specific T and B cells. As for the early pro-inflammatory response, DC activation is triggered by PRR signals. These signals convert resting DCs into potent antigen-presenting cells capable of promoting the expansion and effector differentiation of naive pathogen-specific T cells. However, it has been argued that signals from PRRs are not a prerequisite for DC activation and that pro-inflammatory cytokines have the same effect. Although this may appear like an efficient way to expand the number of DCs that initiate adaptive immunity, evidence is accumulating that DCs activated indirectly by inflammatory cytokines are unable to induce functional T-cell responses. Here, we review the differences between PRR-triggered and cytokine-induced DC activation and speculate on a potential role for DCs activated by inflammatory signals in tolerance induction rather than immunity.     

Mesenchymal stem cells attenuated PLGA-induced inflammatory responses by inhibiting host DC maturation and function

The poly lactic-co-glycolic acid (PLGA) bio-scaffold is a biodegradable scaffold commonly used for tissue repair. However, implanted PLGA scaffolds usually cause serious inflammatory responses around grafts. To improve PLGA scaffold-based tissue repair, it is important to control the PLGA-mediated inflammatory responses. Recent evidence indicated that PLGA induce dendritic cell (DC) maturation in vitro, which may initiate host immune responses. In the present study, we explored the modulatory effects of mesenchymal stem cells (MSC) on PLGA-induced DCs (PLGA-DC). We found that mouse MSCs inhibited PLGA-DC dendrite formation, as well as co-stimulatory molecule and pro-inflammatory factor expression. Functionally, MSC-educated PLGA-DCs promoted Th2 and regulatory T cell differentiation but suppressed Th1 and Th17 cell differentiation. Mechanistically, we determined that PLGA elicited DC maturation via inducing phosphorylation of p38/MAPK and ERK/MAPK pathway proteins in DCs. Moreover, MSCs suppressed PLGA-DCs by partially inactivating those pathways. Most importantly, we found that the MSCs were capable of suppressing DC maturation and immune function in vivo. Also, the proportion of mature DCs in the mice that received MSC-PLGA constructs greatly decreased compared with that of their PLGA-film implantation counterparts. Additionally, MSCs co-delivery increased regulatory T and Th2 cells but decreased the Th1 and Th17 cell numbers in the host spleens. Histological analysis showed that MSCs alleviated the inflammatory responses around the grafted PLGA scaffolds. In summary, our findings reveal a novel function for MSCs in suppressing PLGA-induced host inflammatory response and suggest that DCs are a new cellular target in improving PLGA scaffold-based tissue repair.     

Trypanosoma cruzi calreticulin: In vitro modulation of key immunogenic markers of both canine tumors and relevant immune competent cells

Recombinant calreticulin from Trypanosoma cruzi (rTcCalr), the parasite responsible for Chagas’ disease, binds to Canine Transmissible Venereal Tumor (CTVT) cells from primary cultures and to a canine mammary carcinoma cell line. A Complement-binding assay indicated that interaction of the first component C1q with these tumor cells operated independently of the rTcCalr-presence. This apparent independence could be explained by the important structural similarities that exist among rTcCarl, endogenous normal canine and/or mutated calreticulins present in several types of cancer. In phagocytosis assays, tumor cells treated with rTcCalr were readily engulfed by macrophages and, co-cultured with DCs, accelerated their maturation. In addition, DCs maturation, induced by tumor cells co-cultured with rTcCalr, activated T cells more efficiently than DCs, treated or not with LPS. In an apparent paradox, a decrease in MHC Class I expression was observed when these tumor cells were co-cultivated with rTcCalr. This decrease may be related to a down regulation signaling promoting the rescue of MHC I. Possibly, these in vitro assays may be valid correlates of in vivo sceneries. Based on these results, we propose that rTcCalr improves in vitro the immunogenicity of two widely different tumor cell lines, thus suggesting that the interesting properties of rTcCalr to boost immune responses warrant future studies.     

Optimal stimulation for CD70 induction on human monocyte‐derived dendritic cells and the importance of CD70 in naive CD4+ T‐cell differentiation

Studies in mice have shown that CD70 on dendritic cells (DCs) is sufficient to convert T‐cell tolerance into immunity and hence induce anti‐tumour immune responses. Therefore, it is important to investigate (i) optimal stimuli to induce CD70 on human monocyte‐derived DCs (MoDCs), which are widely used for tumour immunotherapy, and (ii) the role of CD70 in functional differentiation of naive CD4⁺ and CD8⁺ T cells stimulated with MoDCs. We show that interferon‐α (IFN‐α) is a key cytokine to differentiate monocytes into DCs with the capacity to express CD70 upon maturation. CD70 expression on IFN‐α‐induced MoDCs was elicited by different categories of maturation‐inducing factors (Toll‐like receptor ligands, CD40 ligand and pro‐inflammatory mediators), among which prostaglandin E₂ was most effective. Naive T cells stimulated with MoDCs also expressed CD70. Stimulation with MoDCs promoted naive CD4⁺ T cells to acquire the ability to produce T helper type 1 and 2 cytokines in a CD70‐dependent manner. In contrast, the CD70–CD27 interaction diminished the production of an immunoregulatory cytokine IL‐10. The CD27 signal did not play a dominant role in the induction of effector molecules in naive CD8⁺ T cells during the stimulation with MoDCs. This study adds a novel function to the versatile cytokines, type I IFNs, that is, the induction of CD70 on MoDCs. CD70 promotes naive CD4⁺ T cells to acquire immunostimulatory activity through the DC–T‐cell and T‐cell–T‐cell interactions during the stimulation with MoDCs. Hence, the CD70–CD27 interaction may play an important role in inducing effective immune responses in DC‐based immunotherapy.     

Involvement of IL-10 in exhaustion of myeloid dendritic cells and rescue by CD40 stimulation

It has recently been shown that immature dendritic cells (DCs) stimulated by a danger signal undergo transient maturation followed by exhaustion. However, the exact mechanism for this has not been elucidated. In this study, we show that interleukin-10 (IL-10) secreted from transiently matured DCs stimulated by danger signals is responsible for this rapid DC exhaustion. Blocking of the autocrine IL-10 enabled transient mature DCs to maintain the mature phenotype for several days. However, these DCs remained phenotypically unstable because the addition of IL-10 altered the transient mature DCs to exhausted DCs. More importantly, stimulation of DCs by CD40 protected transient mature DCs from IL-10-dependent exhaustion, with the result that mature DCs remained stable in the presence of IL-10. Furthermore, in vivo administration of stable mature DCs pulsed with ovalbumin protein induced antigen-specific cytotoxic T lymphocytes (CTLs) effectively, whereas neither exhausted DCs nor transient mature DCs were able to prime a strong antigen-specific CTL response. These results indicate that DC-T cell engagement via CD40-CD154 is required for stable DC maturation leading to effective CTL induction. Otherwise, DCs stimulated solely by a danger signal are temporarily activated, but then rapidly lose their immune-activating capacity under the influence of autocrine IL-10.     

Enhanced in vitro stimulation of rhesus macaque dendritic cells for activation of SIV-specific T cell responses

The macaque-simian immunodeficiency virus (SIV) system is one of the best animal models available to study the role of dendritic cells (DCs) in transmission and pathogenesis of HIV, as well as to test DC-based vaccine and therapeutic strategies. To better define and optimize this system, the responsiveness of macaque monocyte-derived DCs to a variety of maturation stimuli was examined. Characteristic immunophenotypic and functional DC maturation induced by standard monocyte conditioned medium (MCM) was compared to the activation induced by a panel of stimuli including soluble CD40L, LPS, Poly I:C, PGE₂/TNF, and a cocktail mixture of PGE₂/TNF/IL-1β/IL-6. Immunophenotypic analysis confirmed that all stimuli induced stable up-regulation of CD25, CD40, CD80, CD83, CD86, HLA-DR, DC-LAMP (CD208), and DEC-205 (CD205). In general, macaque DCs exhibited weaker responses to LPS and Poly I:C than human DCs, and soluble CD40L stimulation induced variable expression of CD25. Interestingly, while the endocytic capacity of CD40L-matured cells was down-modulated comparably to DCs matured with MCM or the cocktail, the T cell stimulatory activity was not enhanced to the same extent. The particularly reproducible and potent T cell stimulatory capacity of cocktail-treated DCs correlated with a more homogenous mature DC phenotype, consistently high levels of IL-12 production, and better viability upon reculture compared to DCs activated by other stimuli. Furthermore, cocktail-matured DCs efficiently captured and presented inactivated SIV to SIV-primed T cells in vitro. Thus, the cocktail represents a particularly potent and useful stimulus for the generation of efficacious immunostimulatory macaque DCs.     

两种白血病细胞抗原负载DC的体外诱导特异性CTL应答的比较

目的: 比较树突状细胞 (DC)与白血病细胞融合及白血病细胞冻融抗原负载DC两种白血病DC疫苗诱导抗原特异性CTL应答的能力。方法: 采用羟乙基淀粉 Ficoll两步法分离外周血单个核细胞(PBMC), 通过贴壁 2h获得黏附的单核细胞, 用GM- CSF加IL- 4诱导培养 5d收获细胞。将细胞分为 4组: A组将K562细胞或原代慢性粒细胞白血病(CML)细胞在 500g/LPEG 100mL/LDMSO诱导下与DC融合; B组加入相应细胞数量的白血病细胞冻融抗原; C组将K562细胞或CML细胞与DC共培养; D组: 单独DC培养组。融合前以红色荧光染料PKH26标记K562细胞, 采用流式细胞仪(FCM)检测PKH26 /FITC 抗HLA ABC抗体双标细胞并评估融合率。培养的第 6天, 加入TNF -α诱导DC成熟, 然后分别与自体T细胞共培养, 用MTT比色法检测各组CTL对靶细胞的杀伤活性。结果: GM- CSF加IL -4和TNF- α依次诱导成熟的DC具有经典的DC的形态和表型特征, 在PEG -DMSO的介导下, DC与白血病细胞的融合率为 ( 17. 33 ~29. 94 )%。两种抗原负载方案激活的CTL均对表达K562抗原的细胞具有特异性的细胞毒作用。在效靶比相同时, 融合组诱导CTL的杀伤活性强于冻融抗原致敏DC组。结论: 与冻融抗原致敏的DC相比, DC与白血病细胞融合细胞递呈抗原的效率更高, 体外诱导的CTL特异性杀伤靶     

Persistence of apoptosis-resistant T cell-activating dendritic cells promotes T helper type-2 response and IgE antibody production

Antigen-specific T cell-mediated apoptosis of dendritic cells (DCs) represents a unique down-regulatory mechanism that prevents the continuous activation of T cells by antigen-loaded DCs; this regulatory mechanism is impaired in allergy and as a consequence a large proportion of DCs tends to escape apoptosis following cognate interaction with CD4(+) T cells. However, the biological relevance of greater numbers of apoptosis-resistant DCs to the development of allergic IgE-mediated reactions remained to be determined. Here, we sought to investigate the in vitro and in vivo regulatory features of apoptosis-resistant DCs and to assess their role in host sensitization. Freshly isolated CD11c(+/hi)B220(-)DCs from ovalbumin (OVA)-sensitized, OVA-immunized and na?ve Balb/c mice were cultured with OVA-specific T cells and levels of T cell-mediated DCs apoptosis assessed by flow cytometry. Surviving apoptosis-resistant DCs were then recovered and subsequently co-cultured with OVA-specific CD62L(hi)CD44(low) na?ve T cells or passively transferred into naive syngenic recipients. In vitro profile of DC and T cell lymphokine production, chemokine receptors expression and in vivo, post-adoptive DC transfer T helper (T(H)) and IgE responses were assessed. Apoptosis-resistant DCs showed differential regulatory properties compared to their freshly isolated counterpart independent of the sensitization status of the donor. When co-cultured with na?ve OVA-specific T cells, apoptosis-resistant DCs from either sensitized or immunized mice induced T cells that produced increased levels of IL-4 and reduced levels of IFN-gamma and showed increased expression of T(H)-2 related CCR4 and CCR8 chemokine receptors. Finally, adoptive transfer of apoptosis-resistant DCs, induced higher levels of OVA-specific IgE responses in absence of antigen challenge in syngenic recipients compared to freshly isolated DCs from both sensitized and immunized mice. These data would suggest that sensitization-associated increased numbers of apoptosis-resistant T cell-activating DCs contribute to the generation/maintenance of IgE-mediated allergic reactions.