Bacterial DNA and immunostimulatory CpG oligonucleotides trigger maturation and activation of murine dendritic cells

Bacterial DNA and immunostimulatory (i.s.) synthetic CpG-oligodeoxynucleotides (ODN) act as adjuvants for Th1 responses and cytotoxic T cell responses to proteinaceous antigens. Dendritic cells (DC) can be referred to as “nature's adjuvant” since they display the unique capacity to sensitize naive T cells. Here, we demonstrate that bacterial DNA or i.s. CpG-ODN cause simultaneous maturation of immature DC and activation of mature DC to produce cytokines. These events are associated with the acquisition of professional antigen-presenting cell (APC) function. Unfractionated murine bone marrow-derived DC and FACS^®-fractionated MHC class II^low (termed immature DC) or MHC class II^high populations (termed mature DC) were stimulated with bacterial DNA or i.s. CpG-ODN. Similar to lipopolysaccharide, i.s. CpG-ODN caused up-regulation of MHC class II, CD40 and CD86, but not CD80 on immature and mature DC. In parallel both DC subsets were activated to produce large amounts of IL-12, IL-6 and TNF-α. CpG-ODN-activated DC displayed professional APC function in allogeneic mixed lymphocyte reaction and in staphylococcal enterotoxin B-driven naive T cell responses. We interpret these findings to mean that bacterial DNA and i.s. CpG-ODN cause maturation (first step) and activation (second step) of DC to bring about conversion of immature DC into professional APC.     

The role of antigen-presenting cells and interleukin-12 in the priming of antigen-specific CD4+ T cells by immune stimulating complexes

Immune stimulating complexes (ISCOMs) containing the saponin adjuvant Quil A are vaccine adjuvants that promote a wide range of immune responses in vivo, including delayed-type hypersensitivity (DTH) and the secretion of both T helper 1 (Th1) and Th2 cytokines. However, the antigen-presenting cell (APC) responsible for the induction of these responses has not been characterized. Here we have investigated the role of dendritic cells (DC), macrophages (Mφ) and B cells in the priming of antigen-specific CD4⁺ T cells in vitro by ISCOMs containing ovalbumin (OVA). OVA ISCOMs pulsed bone marrow (BM)-derived DC but not BM Mφ, nor naïve B cells prime resting antigen-specific CD4⁺ T cells, and this response is greatly enhanced if DC are activated with lipopolysaccharide (LPS). Of the APC found in the spleen, only DC had the capacity to prime resting antigen specific CD4⁺ T cells following exposure to OVA ISCOMs in vitro, while Mφ and B cells were ineffective. DC, but not B cells purified from the draining lymph nodes of mice immunized with OVA ISCOMs also primed resting antigen-specific CD4⁺ T cells in vitro, suggesting that DC are also critical in vivo. Using DC and T cells from interleukin (IL)-12 p40^−/− mice, we also identified a crucial role for IL-12 in the priming of optimal CD4⁺ T cell responses by OVA ISCOMs. We suggest that DC are the principal APC responsible for the priming of CD4⁺ T cells by ISCOMs in vivo and that directed targeting of these vectors to DC may enhance their efficancy as vaccine adjuvants.     

MCS-18, a novel natural product isolated from Helleborus purpurascens, inhibits dendritic cell activation and prevents autoimmunity as shown in vivo using the EAE model

Here we report for the first time that MCS-18, a novel natural product isolated from Helleborus purpurascens, is able to inhibit the expression of typical molecules of mature dendritic cells (DC) such as CD80, CD86, and especially of CD83 subsequently leading to a clear and dose-dependent inhibition of the DC-mediated T-cell stimulation. Furthermore, MCS-18 impeded the formation of the typical DC/T-cell clusters, which are essential to induce potent immune responses. Interestingly, MCS-18 also inhibited CCR7 expression on DC which subsequently lead to a dose-dependent block of the CCL19-mediated DC migration. MCS-18 not only inhibited the DC-mediated T-cell stimulation but also the anti-CD3/anti-CD28-mediated T-cell stimulation. Strikingly, MCS-18 also strongly reduced the paralysis associated with the experimental autoimmune encephalomyelitis (EAE), which is a murine model for human multiple sclerosis, in a prophylactic as well as in a “real” therapeutic setting. Even when the EAE was induced for a second time, the MCS-18-treated animals were still protected, suggesting that MCS-18 induces a long-lasting suppressive effect. In addition, and very important for the potential practical application in humans, MCS-18 was also active when administered orally. MCS-18 treatment almost completely reduced leukocyte infiltration in the brain and in the spinal cord. In conclusion, using in vitro as well in vivo assays we were able to show that MCS-18 exerts a strong immunosuppressive activity with remarkable potential for the therapy of diseases characterized by a pathologically over-activated immune system.     

Glucocorticoids down-regulate dendritic cell function in vitro and in vivo

Exogenous glucocorticoid hormones are widely used as therapeutical agents, whereas endogenous glucocorticoids may act as physiological immunosuppressants involved in the control of immune and inflammatory responses. The optimal activation of T lymphocytes requires two distinct signals: the major histocompatibility complex-restricted presentation of the antigen and an additional co-stimulatory signal provided by the antigen-presenting cells. There is ample evidence that, among the cells able to present the antigen, the dendritic cells (DC) have the unique property to activate antigen-specific, naive T cells in vitro and in vivo, and are therefore required for the induction of primary immune responses. In this work, we tested whether glucocorticoids affected the capacity of DC to sensitize naive T cells. Our data show that, in vitro, the steroid hormone analog dexamethasone (Dex) affects the viability of DC, selectively downregulates the expression of co-stimulatory molecules on viable DC, and strongly reduces their immunostimulatory properties. In vivo, a single injection of Dex results in impaired antigen presenting function, a finding which correlates with reduced numbers of splenic DC. These results show that glucocorticoids regulate DC maturation and immune function in vitro and in vivo and suggest that this mechanism may play a role in preventing overstimulation of the immune system.     

Curcumin induces maturation-arrested dendritic cells that expand regulatory T cells in vitro and in vivo

Dendritic cells (DC) and regulatory T cells (T_regs) are vital to the development of transplant tolerance. Curcumin is a novel biological agent extracted from Curcuma longa (turmeric), with anti‐inflammatory and anti‐oxidant activity mediated via nuclear factor (NF)‐κB inhibition. We investigated the immunomodulatory effects of curcumin on human monocyte‐derived and murine DC. Human monocyte‐derived DC (hu‐Mo‐DC) were generated in the presence (CurcDC) or absence (matDC) of 25 µM curcumin, and matured using lipopolysaccharide (1 µg/ml). DC phenotype and allostimulatory capacity was assessed. CD11c⁺ DC were isolated from C57BL/6 mice, pretreated with curcumin and injected into BALB/c mice, followed by evaluation of in vivo T cell populations and alloproliferative response. Curcumin induced DC differentiation towards maturation‐arrest. CurcDC demonstrated minimal CD83 expression (<2%), down‐regulation of CD80 and CD86 (50% and 30%, respectively) and reduction (10%) in both major histocompatibility complex (MHC) class II and CD40 expression compared to matDC. CurcDC also displayed decreased RelB and interleukin (IL)‐12 mRNA and protein expression. Functionally, CurcDC allostimulatory capacity was decreased by up to 60% (P < 0·001) and intracellular interferon (IFN‐γ) expression in the responding T cell population were reduced by 50% (P < 0·05). T cell hyporesponsiveness was due to generation of CD4⁺CD25^hiCD127^loforkhead box P3 (FoxP3)⁺ T_regs that exerted suppressive functions on naïve syngeneic T cells, although the effect was not antigen‐specific. In mice, in vivo infusion of allogeneic CurcDC promoted development of FoxP3⁺ T_regs and reduced subsequent alloproliferative capacity. Curcumin arrests maturation of DC and induces a tolerogenic phenotype that subsequently promotes functional FoxP3⁺ T_regsin vitro and in vivo.     

Wild-type coxsackievirus infection dramatically alters the abundance, heterogeneity, and immunostimulatory capacity of conventional dendritic cells in vivo

In vitro studies have shown that enteroviruses employ strategies that may impair the ability of DCs to trigger T cell immunity, but it is unclear how these viruses affect DCs in vivo. Here, we evaluate the effects of wild-type (wt) coxsackievirus B3 on DCs in vitro and in a murine model in vivo. Although CVB3 does not productively infect the vast majority of DCs, virus infection profoundly reduces splenic conventional DC numbers and diminishes their capacity to prime naïve CD8⁺ T cells in vitro. In contrast to recombinant CVB3, highly pathogenic wt virus infection significantly diminishes the host's capacity to mount T cell responses, which is temporally associated with the loss of CD8α⁺ DCs. Our findings demonstrate that enterovirus infection substantially alters the number, heterogeneity, and stimulatory capacity of DCs in vivo, and these dramatic immunomodulatory effects may weaken the host's capacity to mount antiviral T cell responses.     

Dichotomy between<Emphasis Type="Italic"> Lactobacillus rhamnosus</Emphasis> and<Emphasis Type="Italic"> Klebsiella pneumoniae</Emphasis> on dendritic cell phenotype and function

The reaction of the intestinal immune system to intestinal bacteria shows striking differences between various bacterial strains. Whereas Klebsiella pneumoniae induces a fierce proinflammatory reaction, the probiotic strain Lactobacillus rhamnosus has clear anti-inflammatory effect in gastrointestinal disease and allergy. The molecular basis for this dichotomy is poorly understood but is likely to involve different modulation of antigen-presenting dendritic cells (DC) by L. rhamnosus and K. pneumoniae. Hence we evaluated phenotypic and functional characteristics of DC matured in the presence of L. rhamnosus and K. pneumoniae. Monocyte-derived immature DC were cultured in the presence of live bacteria to obtain mature DC. Both micro-organisms induced maturation of immature DC as shown by CD83 and CD86 expression, but receptors involved in activation of Th1 cells were expressed predominantly on DC exposed to K. pneumoniae. In contrast to K. pneumoniae, maturation with L. rhamnosus resulted in lower TNF-, IL-6, and IL-8 production by immature DC and lower IL-12 and IL-18 production by mature DC. Moreover, L. rhamnosus led to the development of T cells without a typical Th phenotype whereas K. pneumoniae induced a Th1 immune response, dependent mainly on IL-12 production. Thus our results strongly support the concept that differential modulation of DC explains the differences in the immune response to various bacterial strains and indicates that K. pneumoniae induces Th1 immune responses via DC.Abbreviations DC Dendritic cell - FACS Fluorescence-activated cell sorter - ICAM Intercellular adhesion molecule - IFN Interferon - IL Interleukin - LPS Lipopolysaccharide - MFI Mean fluorescence intensity - PAMP Pathogen associated molecular pattern - Th T helper cell - TNF Tumor necrosis factor     

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.     

Role of Dendritic Cells in the Pathogenesis of Whipple's Disease

Accumulation of Tropheryma whipplei-stuffed macrophages in the duodenum, impaired T. whipplei-specific Th1 responses, and weak secretion of interleukin-12 (IL-12) are hallmarks of classical Whipple''s disease (CWD). This study addresses dendritic cell (DC) functionality during CWD. We documented composition, distribution, and functionality of DC ex vivo or after in vitro maturation by fluorescence-activated cell sorting (FACS) and by immunohistochemistry in situ. A decrease in peripheral DC of untreated CWD patients compared to healthy donors was due to reduced CD11c^high myeloid DC (M-DC). Decreased maturation markers CD83, CD86, and CCR7, as well as low IL-12 production in response to stimulation, disclosed an immature M-DC phenotype. In vitro-generated monocyte-derived DC from CWD patients showed normal maturation and T cell-stimulatory capacity under proinflammatory conditions but produced less IL-12 and failed to activate T. whipplei-specific Th1 cells. In duodenal and lymphoid tissues, T. whipplei was found within immature DC-SIGN⁺ DC. DC and proliferating lymphocytes were reduced in lymph nodes of CWD patients compared to levels in controls. Our results indicate that dysfunctional IL-12 production by DC provides suboptimal conditions for priming of T. whipplei-specific T cells during CWD and that immature DC carrying T. whipplei contribute to the dissemination of the bacterium.     

Contribution of dendritic cells to measles virus induced immunosuppression

Measles virus (MV) remains an important pathogen in children worldwide. The morbidity and mortality of MV is associated with severe immune suppression. Dendritic cells (DCs) were identified as initial target cells in vivo, and DCs were efficiently infected by MV in vitro. MV infection of DCs likely contributes to functional deficiency in these cells; therefore playing a role in MV‐induced immunosuppression. DCs appeared to mature phenotypically; however, the ability of infected cells to stimulate T cells was compromised. Phenotypic maturation of infected immature DCs was partially controlled by IFN production; however, infected DCs also maintained markers of an immature phenotype such as the continued uptake of antigen and lack of expression of chemokine receptor CCR7. Furthermore, mature DCs did not appear to maintain phenotypic maturation following infection demonstrated by decreased MHC and co‐stimulatory molecule expression. Several mechanisms of MV‐induced DC dysfunction have been suggested, each likely contributing to the immunosuppressive effect of MV‐infected DCs. Infected DCs responded aberrantly to secondary maturation stimuli such as CD40L or TLR4 stimulation. MV infection resulted in apoptosis in DC/T‐cell cocultures, which may contribute to a reduced T‐cell response. Additionally, the immunological synapse between infected DCs and T cells was compromised resulting in reduced T‐cell interaction times and activation signaling. The mechanisms of MV contribution to DC dysfunction appear multifaceted and central to MV‐induced immunosuppression. Published 2012. This article is a U.S. Government work and is in the public domain in the USA.     

Anti-P-selectin lectin-EGF domain monoclonal antibody inhibits the maturation of human immature dendritic cells

Dendritic cells (DCs) are professional antigen-presenting cells with the ability to initiate primary T cell responses. While it is well known that inflammatory stimuli induce the functional maturation of immature DCs, whether adhesion molecule selectins regulate DC maturation is poorly understood. Using anti-P-selectin lectin-EGF domain monoclonal antibody (PsL-EGFmAb) that blocks the adhesion of P-, E-, and L-selectin, we demonstrate herein that selectins play important role in stimulating functional maturation of immature DCs. Immature DCs are generated from human cord blood CD34+ hematopoietic stem/progenitor cells that were cultured in the presence of stem cell factor, Fms-like tyrosine-kinase-3 ligand, granulocyte-macrophage colony stimulating factor, and transform growth factor-beta1. When stimulated with tumor necrosis factor-alpha (TNF-alpha), immature DCs differentiated into mature DCs, producing increased levels of costimulatory molecules and interleukin (IL)-12 and obtaining the ability to potently activate na?ve T cells. Interestingly, in contrast to mature DCs derived from TNF-alpha-induced immature DC cultures without PsL-EGFmAb, immature DCs treated with PsL-EGFmAb for 7 days were completely blocked their maturation, as evidenced by decreased expression of costimulatory molecules CD80, CD86, and CD83, inhibited production of IL-12, and inability to activate na?ve T cells in vitro. Thus, blockade of selectins using PsL-EGFmAb will prove to be a valuable tool for the study of the molecular mechanisms of DC maturation, as well as for the prevention and treatment of DC-mediated autoimmunity.     

Phenotypic and functional characterization of CD11c+ dendritic cell population in mouse Peyer's patches

The antigen-presenting cell system in the gastrointestinal tract, one of three main sites (skin and lung being the others) of primary antigen contact, is poorly understood. Our study focused on dendritic cells (DC) as possible candidates for antigen uptake, processing and presentation in mucosal inductive sites, such as Peyer's patches (PP). To investigate the morphology, immunophenotype and stimulatory activity of intestinal DC, a procedure was developed to obtain a cell population by using collagenase digestion of PP, density centrifugation and cell sorting on the basis of CD11c expression. The resultant low-density cell fraction consisted of a nonadherent cell population expressing different intensities of CD11c that could at least be characterized by typical DC morphology (e.g. abundant cytoplasma with veil-like cytoplasmatic dendrites, irregularly shaped nuclei, multivesicular and multilamellar bodies), constitutive levels of surface MHC class II, the presence of macrophage-specific markers, such as F4/80, Mac-I and Fc receptors, respectively, on subpopulations of CD11c⁺ sorted cells and expression of adhesion and co-stimulatory receptors like ICAM-1 and CD44. The capability of this low-density CD11c⁺ fraction to stimulate T cell responses was demonstrated in primary allogeneic mixed-lymphocyte reactions (MLR). Herein, we show that the freshly isolated CD11c⁺ cells showed weak accessory function, but develop this capacity following short-term culture in vitro in the presence of granulocyte/macrophage colony-stimulating factor. Although the nature and functional capacity of the isolated CD11c⁺ needs further clarification, these preliminary results describing phenotype and accessory function provide some evidence that these cells isolated from the PP may be immature forms of DC and play a crucial role as antigen-presenting cells with important implications for understanding the complex network regulating intestinal antigen uptake, processing and presentation.     

Bruton's tyrosine kinase defect in dendritic cells from X-linked agammaglobulinaemia patients does not influence their differentiation,maturation and antigen-presenting cell function

X‐linked agammaglobulinaemia (XLA) is a primary immunodeficiency disease characterized by very low levels or even absence of circulating antibodies. The immunological defect is caused by deletions or mutations of Bruton's tyrosine kinase gene (Btk), whose product is critically involved in the maturation of pre‐B lymphocytes into mature B cells. Btk is expressed not only in B lymphocytes but also in cells of the myeloid lineage, including dendritic cells (DC). These cells are professional antigen presenting cells (APC) that play a fundamental role in the induction and regulation of T‐cell responses. In this study, we analysed differentiation, maturation, and antigen‐presenting function of DC derived from XLA patients (XLA‐DC) as compared to DC from age‐matched healthy subjects (healthy‐DC). We found that XLA‐DC normally differentiate from monocyte precursors and mature in response to lipopolysaccharide (LPS) as assessed by de novo expression of CD83, up‐regulation of MHC class II, B7·1 and B7·2 molecules as well as interleukin (IL)‐12 and IL‐10 production. In addition, we demonstrated that LPS stimulated XLA‐DC acquire the ability to prime naïve T cells and to polarize them toward a Th1 phenotype, as observed in DC from healthy donors stimulated in the same conditions. In conclusion, these data indicate that Btk defect is not involved in DC differentiation and maturation, and that XLA‐DC can act as fully competent antigen presenting cells in T cell‐mediated immune responses.     

Tolerance induced via TLR2 and TLR4 in human dendritic cells: role of IRAK-1

Background

Modified Vaccinia Ankara (MVA) is a highly attenuated strain of vaccinia virus (VV) that has lost approximately 15% of the VV genome, along with the ability to replicate in most mammalian cells. It has demonstrated impressive safety and immunogenicity profile in both preclinical and clinical studies, and is being actively explored as a promising vaccine vector for a number of infectious diseases and malignancies. However, little is known about how MVA interacts with the host immune system constituents, especially dendritic cells (DCs), to induce strong immune responses despite its inability to replicate in vivo. Using in vitro and in vivo murine models, we systematically investigated the susceptibility of murine DCs to MVA infection, and the immunological consequences of the infection.

Results

Our data demonstrate that MVA preferentially infects professional antigen presenting cells, especially DCs, among all the subsets of hematolymphoid cells. In contrast to the reported blockage of DC maturation and function upon VV infection, DCs infected by MVA undergo phenotypic maturation and produce innate cytokine IFN-α within 18 h of infection. Substantial apoptosis of MVA-infected DCs occurs after 12 h following infection and the apoptotic DCs are readily phagocytosed by uninfected DCs. Using MHC class I – deficient mice, we showed that both direct and cross-presentation of viral Ags are likely to be involved in generating viral-specific CD8⁺ T cell responses. Finally, DC depletion abrogated the T cell activation in vivo.

Conclusion

We present the first in vivo evidence that among hematolymphoid cells, DCs are the most susceptible targets for MVA infection, and DC-mediated Ag presentation is required for the induction of MVA-specific immune responses. These results provide important information concerning the mechanisms by which strong immune responses are elicited to MVA-encoded antigens and may inform efforts to further improve the immunogenicity of this already promising vaccine vector.     

Establishment of a cell line with features of early dendritic cell precursors from fetal mouse skin

During ontogeny, the skin is progressively populated by major histocompatibility complex class II-negative dendritic cell (DC) precursors that then mature into efficient antigen-presenting cells (APC). To characterize these DC progenitors better, we generated myeloid cell lines from fetal mouse skin by infecting cell suspensions with a retroviral vector carrying an env^AKR-myc^MH2 fusion gene. These cells, represented by the line FSDC, displayed a dendritic morphology and their proliferation in serum-free medium was promoted by granulocyte/macrophage colony-stimulating factor (GM-CSF), but not macrophage-CSF. FSDC expressed strong surface-membrane ATP/ADPase activity, intracellular staining for 2A1 antigen, and a surface phenotype consistent with a myeloid precursor: H-2^d.b+, I-A^d.b+, CD54⁺, CD11b⁺, CD11c⁺, 2.4G2⁺, F4/80⁺, CD44⁺, 2F8⁺, ER-MP 12^?, Sca-1⁺, Sca-2⁺, NLDC-145^?, B7.2⁺, B7.1^?, J11d^?, B220^?, Thy-1^?, and CD3^?. FSDC stimulated poorly allogeneic or syngeneic T cells in the primary mixed-leukocyte reaction, and markedly increased this function after treatment with GM-CSF, GM-CSF and interleukin (IL)-4 or interferon-γ (IFN-γ); in contrast, stem cell factor, IL-1α and tumor necrosis factor-α had no effect. Preculture with IFN-γ was required for presentation of haptens to primed T cells in vitro. However, FSDC, even after cytokine activation, were less potent APC than adult epidermal Langerhans cells in both of the above assays. Finally, FSDC derivatized with haptens and injected either intravenously or subcutaneously could efficiently induce contact sensitivity responses in naive syngeneic mice. The results indicate that fetal mouse skin is colonized by myeloid precursors possessing a macrophage/immature DC-like surface phenotype and priming capacity in vivo. These cells need further differentiation and activation signals (e.g. cytokines) to express their antigen presenting potential in vitro.     

Induced regulatory T cells are phenotypically unstable and do not protect mice from rapidly progressive glomerulonephritis

Regulatory T (Treg) cells are a suppressive CD4⁺ T‐cell subset. We generated induced Treg (iTreg) cells and explored their therapeutic potential in a murine model of rapidly progressive glomerulonephritis. Polyclonal naive CD4⁺ T cells were cultured in vitro with interleukin‐2 (IL‐2), transforming growth factor‐β1, all‐trans‐retinoic acid and monoclonal antibodies against interferon‐γ and IL‐4, generating Foxp3⁺ iTreg cells. To enhance their suppressive phenotype, iTreg cultures were modified with the addition of a monoclonal antibody against IL‐12p40 or by using RORγt^–/– CD4⁺ T cells. Induced Treg cells were transferred into models of delayed‐type hypersensitivity and experimental glomerulonephritis. The iTreg cells exhibited comparable surface receptor expression and in vitro suppressive ability to natural Treg cells, but did not regulate antigen‐specific delayed‐type hypersensitivity or systemic inflammatory immune responses, losing Foxp3 expression in vivo. In glomerulonephritis, transferred iTreg cells did not prevent renal injury or modulate systemic T helper type 1 immune responses. Induced Treg cells cultured with anti‐IL‐12p40 had an enhanced suppressive phenotype in vitro and regulated dermal delayed‐type hypersensitivity in vivo, but were not protective against renal injury, losing Foxp3 expression, especially in the transferred cells recruited to the kidney. Use of RORγt^–/– CD4⁺ T cells or iTreg cells generated from sensitized CD4⁺ Foxp3^– cells did not regulate renal or systemic inflammatory responses in vivo. In conclusion, iTreg cells suppress T‐cell proliferation in vitro, but do not regulate experimental glomerulonephritis, being unstable in this inflammatory milieu in vivo.     

Dendritic cells pulsed with keyhole limpet hemocyanin and cryopreserved maintain anti-tumor activity in a murine melanoma model

We compared viability, phenotype, in vitro function and therapeutic efficacy of murine unpulsed-dendritic cells (-DC), DC pulsed with keyhole limpet hemocyanin (KLH-DC) and cryopreserved KLH-DC (C-KLH-DC). Mean viability (% ± SE) of unpulsed-DC, KLH-DC and C-KLH-DC was 93.6 ± 0.9, 93.9 ± 0.8 and 87.4 ± 1.6, respectively. Pulsing DC with KLH did not induce maturation or affect in vitro function. Cryopreservation of KLH-DC reduced MHC I, CD80 and CD86 expression, endocytic capacity and allogeneic splenocyte stimulatory capacity. Intratumoral (i.t.) vaccination of mice bearing s.c. D5 melanoma with unpulsed-DC, KLH-DC or C-KLH-DC elicited comparable anti-tumor immune responses and inhibited tumor growth to the same extent. Combining radiotherapy with i.t. unpulsed-DC, KLH-DC or C-KLH-DC administration enhanced induction of anti-tumor immune responses and inhibition of tumor growth to a similar degree. Cryopreservation of KLH-DC slightly reduces viability, expression of co-stimulatory cell surface markers and in vitro function; however, in vivo anti-tumor activity is fully maintained with or without radiotherapy.     

Interleukin-7 promotes the survival of human CD4+ effector/memory T cells by up-regulating Bcl-2 proteins and activating the JAK/STAT signalling pathway

Major histocompatibility complex (MHC) class I‐specific inhibitory natural killer receptors (iNKRs) are expressed by subsets of T cells but the mechanisms inducing their expression are poorly understood, particularly for killer‐cell immunoglobulin‐like receptors (KIRs). The iNKRs are virtually absent from the surface of cord blood T cells but we found that KIR expression could be induced upon interleukin‐2 stimulation in vitro. In addition, KIR expression was enhanced after treatment with 5‐aza‐2′‐deoxycytidine, suggesting a role for DNA methylation. In vivo induction of KIR expression on cord blood T cells was also observed during a human congenital infection with Trypanosoma cruzi which triggers activation of fetal CD8⁺ T cells. These KIR⁺ T cells had an effector and effector/memory phenotype suggesting that KIR expression was consecutive to the antigenic stimulation; however, KIR was not preferentially found on parasite‐specific CD8⁺ T cells secreting interferon‐γ upon in vitro restimulation with live T. cruzi. These findings show that KIR expression is likely regulated by epigenetic mechanisms that occur during the maturation process of cord blood T cells. Our data provide a molecular basis for the appearance of KIRs on T cells with age and they have implications for T‐cell homeostasis and the regulation of T‐cell‐mediated immune responses.     

Thymic Dendritic Cells: Phenotype and Function

Interdigitating (IDC) cells of the thymus have been characterized in situ by their ultrastructure and phenotype. Thymic dendritic cells (DC), thought to represent their in vitro correlate, resemble splenic DC in their ability to initiate peripheral T cell responses. In vivo, however, DC of the thymus have been implicated in tolerance induction, although at one time they were thought to impart MHC-restriction on developing T cells. Our present understanding of these areas is reviewed here.

An in vitro model has been developed to address directly the function of DC in the thymus. Mature DC and immature thymocytes migrate into deoxyguanosine-treated thymus lobes where they adopt a reciprocal distribution, DC homing primarily to the medulla while the thymocytes remain in the cortex. These observations support the close relationship between thymic DC and IDC and provide a powerful tool to examine the role of DC in thymocyte ontogeny.     

HIV I Infection of Dendritic Cells

Dendritic cells (DC) from human peripheral blood are susceptible to productive and probably to latent infection with HIV-I [18, 29]. Infection of DC also occurs in vivo since in HIV-seropositive individuals Langerhans’ cells of the skin [16] and DC from peripheral blood ([17], in preparation) are infected. In peripheral blood 3–25% of DC, identified as large, low-density cells lacking monocyte markers, are infected as judged by in situ hybridization with an HIV probe. This contrasts with the lower proportion (<0.2%) of other cells infected. DC exposed to HIV in vitro or in vivo fail to present other antigens or mitogens to stimulate T cells [29, 38, 41]. This functional defect in infected DC is not blocked by the presence of soluble CD4 antigen and occurs in the absence of T cell infection suggesting a block at the level of the antigen-presenting cell itself. Infection, depletion and dysfunction of DC in HIV seropositive patients is already present in asymptomatic individuals and this precedes the appearance of T cell defects. We speculate that loss of functional DC may be a fundamental defect leading to a block in recruitment of resting T cells into immune responses.

In contrast to the HIV-induced impairment of antigen presentation by DC, these cells were potent stimulators of responses to the HIV antigens themselves. Normal DC infected with HIV in vitro stimulated primary proliferative and cytotoxic T cell responses ([52], in preparation). These were produced in cells from individuals expressing a range of different MHC types but the cytotoxic cells, once produced, killed autologous but not allogeneic, infected T cell blasts. Primary response to viral peptides can also be produced suggesting that this system may be useful for identifying immunogenic epitopes of HIV using cells from sero-negative, non-immunocompromised individuals.