Altered phenotype and function of blood dendritic cells in multiple sclerosis are modulated by IFN-beta and IL-10.

Multiple sclerosis (MS) is assumed to result from autoaggressive T cell-mediated immune responses, in which T helper type 1 (Th1) cells producing cytokines, e.g. IFN-gamma and lymphotoxin promote damage of oligodendrocyte-myelin units. Dendritic cells (DCs) as potent antigen presenting cells initiate and orchestrate immune responses. Whether phenotype and function of DCs with respect to Th1 cell promotion are altered in MS, are not known. This study revealed that blood-derived DCs from MS patients expressed low levels of the costimulatory molecule CD86. In addition, production of IFN-gamma by blood mononuclear cells (MNCs) was strongly enhanced by DCs derived from MS patients. IFN-beta and IL-10 inhibited the costimulatory capacity of DCs in mixed lymphocyte reaction (MLR) and showed additive effects on suppression of IL-12 production by DCs. Correspondingly, DCs pretreated with IFN-beta and IL-10 significantly suppressed IFN-gamma production by MNCs. IFN-beta in vitro also upregulated CD80 and, in particular, CD86 expression on DCs. In vitro, anti-CD80 antibody remarkably increased, while anti-CD86 antibody inhibited DC-induced IL-4 production in MLR. We conclude that DC phenotype and function are altered in MS, implying Th1-biased responses with enhanced capacity to induce Th1 cytokine production. In vitro modification of MS patients' DCs by IFN-beta and IL-10 could represent a novel way of immunomodulation and of possible usefulness for future immunotherapy of MS.     

CD40L-expressing CD8 T cells prime CD8alpha(+) DC for IL-12p70 production

CD8alpha(+) DC are implicated as the principle DC subset for cross-presentation and cross-priming of cytotoxic CD8 T cell responses. In this study, we demonstrate another unique facet of the CD8alpha(+) DC and CD8 T cell relationship, by showing that CD8 T cells reciprocally activate CD8alpha(+) DC, but not CD8alpha(-) DC, for IL-12p70 production, the key Th1-promoting cytokine. This effect was observed during an antigen-specific interaction between DC and activated CD8 T cells, along with secondary TLR stimulation of DC by LPS. Activated CD8 T cells use a combination of IFN-gamma and CD40L, which is rapidly up-regulated post-stimulation, to prime DC for IL-12p70 production during an antigen-specific response. Our results suggest that the interaction between CD8alpha(+) DC and antigen-primed CD8 T cells may form an important component of Th1-mediated immunity through the induction of IL-12p70.     

Engagement of human monocyte-derived dendritic cells into interleukin (IL)-12 producers by IL-1beta + interferon (IFN)-gamma

Dendritic cells (DCs) are potent antigen-presenting cells and can induce tumour- or pathogen-specific T cell responses. For adoptive immunotherapy purposes, immature DCs can be generated from adherent monocytes using granulocyte macrophage colony stimulating factor (GM-CSF) and interleukin (IL)-4, and further maturation is usually achieved by incubation with tumour necrosis factor (TNF)-alpha. However, TNF-alpha-stimulated DCs produce low levels of IL-12. In this study, we compared the effects of TNF-alpha, interferon (IFN)-gamma, IL-1beta or IFN-gamma + IL-1beta on the phenotypic and functional maturation of DCs. Our results show that IFN-gamma, but not IL-1beta, augmented the surface expression of CD80, CD83 and CD86 molecules without inducing IL-12 production from DCs. However, IL-1beta, but not IFN-gamma, induced IL-12 p40 production by DCs without enhancing phenotypic maturation. When combined, IFN-gamma + IL-1beta treatment profoundly up-regulated the expression of CD80, CD83, CD86 and major histocompatibility complex (MHC) class II antigens. Furthermore, IFN-gamma + IL-1beta-treated DCs produced larger amounts of IL-12 and induced stronger T cell proliferation and IFN-gamma secretion in primary allogeneic mixed lymphocyte reaction (MLR) than did TNF-alpha-treated DCs. Our results show that IFN-gamma + IL-1beta induced human monocyte-derived DCs to differentiate into Th1-prone mature DCs.     

Dendritic cells partially abrogate the regulatory activity of CD4+CD25+ T cells present in the human peripheral blood

The factors that influence the functionality of human CD4(+)CD25(+) regulatory T cells are not well understood. We sought to characterize the effects of dendritic cells (DCs) on the in vitro regulatory activity of CD4(+)CD25(+) T cells obtained from peripheral blood of healthy human donors. Flow cytometry showed that a higher proportion of CD4(+)CD25(+(High)) T cells expressed surface glucocorticoid-induced tumor necrosis factor receptor family-related protein (GITR) and CTL-associated antigen 4 than CD4(+)CD25(-) or CD4(+)CD25(+(Med-low)) T cells. Intracellular Foxp3 was equivalently expressed on CD4(+)CD25(+(All)), CD4(+)CD25(+(High)), CD4(+)CD25(+(Med-low)) and CD4(+)CD25(-) T cell populations, irrespective of GITR and CTL-associated antigen 4 expression. CD4(+)CD25(+) T cells were isolated and then cultured in vitro with CD4(+)CD25(-) responder T cells and stimulated with anti-CD3 antibodies, and immature dendritic cells (iDCs), mature dendritic cells (mDCs), PBMCs or PBMCs plus anti-CD28 antibodies to provide co-stimulation. In addition, secretion of the T(h)1 cytokine IFN-gamma, IL-2 and the immunoregulatory cytokines, IL-10 and transforming growth factor (TGF)-beta, were also assessed in these cultures. We found that iDCs and mDCs were capable of reversing the suppression of proliferation mediated by CD4(+)CD25(+) regulatory T cells. However, the reversal of suppression by DCs was not dependent upon the increase of IFN-gamma and IL-2 production or inhibition of IL-10 and/or TGF-beta production. Therefore, DCs are able to reverse the suppressive effect of regulatory T cells independent of cytokine production. These results suggest for the first time that human DCs possess unique abilities which allow them to influence the functions of regulatory T cells in order to provide fine-tuning in the regulation of T cell responses.     

Dendritic cells generated in the presence of GM-CSF plus IL-15 prime potent CD8+ Tc1 responses in vivo

Dendritic cells (DC) comprise a system of professional antigen-presenting cells, which induce the stimulation of very rare antigen-specific naive T cells. DC progenitors can be stimulated to differentiate into immature DC by various growth factors, including GM-CSF and IL-4. Here we show that IL-15, in combination with GM-CSF, is a growth factor for murine DC. Murine bone marrow cells, depleted of T cells, B cells, I-A+ cells and Gr-1+ granulocytes, and cultured in the presence of GM-CSF plus IL-15 (IL-15 DC), yielded DC expressing high levels of CD11c and MHC class II molecules, as well as CD11b. These cells expressed significant levels of CD40, CD80 and CD86, and could stimulate allogeneic CD4+ T cells efficiently. Interestingly, IL-15 DC were far superior to DC generated with GM-CSF plus IL-4 in stimulating allogeneic CD8+ T cells in vitro. Consistent with this, IL-15 DC induced much more potent antigen-specific CD8+ T cell responses with high levels of Th1 cytokines in vivo, compared to DC generated with GM-CSF plus IL-4, or with GM-CSF plus TGF-beta, or with GM-CSF alone. Together, these data suggest that IL-15 promotes the development of DC, which induce potent Th1 and Tc1 responses in vivo. This suggests potential roles for these IL-15 DC cells in the immunotherapy of tumors and infectious diseases.     

Human TSLP directly enhances expansion of CD8+ T cells

Human thymic stromal lymphopoietin (TSLP) promotes CD4(+) T-cell proliferation both directly and indirectly through dendritic cell (DC) activation. Although human TSLP-activated DCs induce CD8(+) T-cell proliferation, it is not clear whether TSLP acts directly on CD8(+) T cells. In this study, we show that human CD8(+) T cells activated by T-cell receptor stimulation expressed TSLP receptor (TSLPR), and that TSLP directly enhanced proliferation of activated CD8(+) T cells. Although non-stimulated human CD8(+) T cells from peripheral blood did not express TSLPR, CD8(+) T cells activated by anti-CD3 plus anti-CD28 did express TSLPR. After T-cell receptor stimulation, TSLP directly enhanced the expansion of activated CD8(+) T cells. Interestingly, using monocyte-derived DCs pulsed with a cytomegalovirus (CMV)-specific pp65 peptide, we found that although interleukin-2 allowed expansion of both CMV-specific and non-specific CD8(+) T cells, TSLP induced expansion of only CMV-specific CD8(+) T cells. These results suggest that human TSLP directly enhances expansion of CD8(+) T cells and that the direct and indirect action of TSLP on expansion of target antigen-specific CD8(+) T cells may be beneficial to adoptive cell transfer immunotherapy.     

Differential capacity of CD8α+ or CD8α− dendritic cell subsets to prime for eosinophilic airway inflammation in the T-helper type 2-prone milieu of the lung

BACKGROUND: Different subsets of dendritic cells (DCs), identified in mouse spleen by their differential expression of CD8 alpha, can induce different T-helper (Th) responses after systemic administration. CD8 alpha(-) DCs have been shown to preferentially induce Th type 2 (Th2) responses whereas CD8 alpha(+) DCs induce Th1 responses. OBJECTIVE: To study if these DC subsets can still induce different Th responses in the Th2-prone milieu of the lung and differentially prime for eosinophilic airway inflammation, typical of asthma. METHODS: Donor mice first received daily Flt3L injections to expand DC numbers. Purified CD8 alpha(+) or CD8 alpha(-) splenic DCs were pulsed with ovalbumin (OVA) or phosphate-buffered saline and injected intratracheally into recipient mice in which carboxyfluorescein diacetate succinimidyl ester-labelled OVA-specific T cell receptor transgenic T cells had been injected intravenously 2 days earlier. T cell proliferation and cytokine production of Ag-specific T cells were evaluated in the mediastinal lymph nodes (MLNs) 4 days later. The capacity of both subsets of DCs, to prime for eosinophilic airway inflammation was determined by challenging the mice with OVA aerosol 10 days later. RESULTS: CD8 alpha(-) DCs migrated to the MLN and induced a vigorous proliferative T cell response accompanied by high-level production of IL-4, IL-5, IL-10 and also IFN-gamma during the primary response and during challenge with aerosol, leading to eosinophilic airway inflammation. In the absence of migration to the MLN, CD8 alpha(+) DCs still induced a proliferative response with identical levels of IFN-gamma but reduced Th2 cytokines compared with CD8 alpha(-) DCs, which led to weak eosinophilic airway inflammation upon OVA aerosol challenge. Unpulsed DCs did not induce proliferation or cytokine production in Ag-specific T cells. CONCLUSION: CD8 alpha(-) DCs are superior compared with CD8 alpha(+) DCs in inducing Th2 responses and eosinophilic airway inflammation in the Th2-prone environment of the lung.     

Tolerogenic dendritic cells pulsed with enterobacterial extract suppress development of colitis in the severe combined immunodeficiency transfer model

Immunomodulatory dendritic cells (DCs) that induce antigen-specific T-cell tolerance upon in vivo adoptive transfer are promising candidates for immunotherapy of autoimmune diseases. The feasibility of such a strategy has recently proved its efficacy in animal models of allotransplantation and experimental allergic encephalitis, but the effect in inflammatory bowel disease has not yet been demonstrated. In severe combined immunodeficient (SCID) mice, adoptively transferred CD4(+) CD25(-) T cells repopulate the lymphoid tissues and lead to development of chronic colitis characterized by CD4(+) T-cell proliferation against enterobacterial extract in vitro. In this model, we adoptively transferred in-vitro-generated bone-marrow-derived DCs exposed to interleukin-10 (IL-10) and an enterobacterial extract. We show that these cells are CD11c positive with intermediate expression of CD40, CD80 and CD86 and have a diminished secretion of IL-6, IL-12 p40/70, tumour necrosis factor-alpha and keratinocyte-derived chemokine (KC) compared to DCs treated with enterobacterial extract alone. In vivo, these cells prevented weight loss in SCID mice adoptively transferred with CD4(+) CD25(-) T cells, resulted in a lower histopathology colitis score and tended to result in higher serum levels of IL-1alpha, IL-10, IL-12, IL-13, IL-17, KC and monokine induced by interferon-gamma (MIG). These data underscore the potential of using immunomodulatory DCs to control inflammatory bowel disease and demonstrate its potential use in future human therapeutic settings.     

GM-CSF-induced, bone-marrow-derived dendritic cells can expand natural Tregs and induce adaptive Tregs by different mechanisms

In our earlier work, we had shown that GM-CSF treatment of CBA/J mice can suppress ongoing thyroiditis by inducing tolerogenic CD8α(-) DCs, which helped expand and/or induce CD4(+)Foxp3(+) Tregs. To identify the primary cell type that was affected by the GM-CSF treatment and understand the mechanism by which Tregs were induced, we compared the effect of GM-CSF on matured spDCs and BMDC precursors in vitro. Matured spDCs exposed to GM-CSF ex vivo induced only a modest increase in the percentage of Foxp3-expressing T cells in cocultures. In contrast, BM cells, when cultured in the presence of GM-CSF, gave rise to a population of CD11c(+)CD11b(Hi)CD8α(-) DCs (BMDCs), which were able to expand Foxp3(+) Tregs upon coculture with CD4(+) T cells. This contact-dependent expansion occurred in the absence of TCR stimulation and was abrogated by OX40L blockage. Additionally, the BMDCs secreted high levels of TGF-β, which was required and sufficient for adaptive differentiation of T cells to Foxp3(+) Tregs, only upon TCR stimulation. These results strongly suggest that the BMDCs differentiated by GM-CSF can expand nTregs and induce adaptive Tregs through different mechanisms.     

Inhibition of human allergic T-cell responses by IL-10-treated dendritic cells: differences from hydrocortisone-treated dendritic cells

BACKGROUND: Dendritic cells (DCs) are able to induce human allergic T(H)1 responses as well as T(H)2 responses. OBJECTIVE: In this study, we examined the effect of antiinflammatory agents such as IL-10 and hydrocortisone (HC) on the accessory function of DCs and the resulting T-cell response, especially that of T(H)2 cells. METHODS: Naive and memory CD4(+) T cells from atopic donors were stimulated with autologous allergen-pulsed DCs generated from CD14(+) monocytes by culture with GM-CSF/IL-4 and fully matured with IL-1 beta, TNF-alpha, and PGE(2) in the presence or absence of IL-10 or HC. RESULTS: IL-10-treated DCs and, to a lesser extent, HC-treated DCs showed a decreased expression of MHC II molecules, the costimulatory molecule CD86, and the DC-specific marker CD83, as well as a strongly reduced IL-12 secretion. Consequently, T-cell proliferation was reduced after stimulation with IL-10- or HC-treated DCs alike. However, pretreatment of DCs with IL-10 inhibited the production of T(H)1 and T(H)2 cytokines by T cells, whereas HC-treated DCs inhibited production of IFN-gamma but induced an increased release of IL-4 and no change in IL-5. Both effects were long-lasting; cytokine production remained low (which was due not to enhanced apoptosis but to functional hyporesponsiveness) or even increased after restimulation with fully matured DCs. CONCLUSION: These data indicate that IL-10- or HC-treated DCs differ in their ability to influence human allergic T-cell responses. This has major implications for therapeutic strategies aiming at the downregulation of proallergic T(H)2 responses.     

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.     

IL-4 is more effective than IL-13 for in vitro differentiation of dendritic cells from peripheral blood mononuclear cells

Dendritic cells (DCs) are the most potent professional antigen-presenting cells which can activate T cells to induce the primary immune response. For clinical studies, DCs are often differentiated in vitro from peripheral blood mononuclear cells (PBMCs) through treatment with granulocyte macrophage colony-stimulating factor (GM-CSF) and IL-4. However, IL-13, a cytokine closely related to IL-4, has also been reported to induce differentiation equally or more efficiently when used with GM-CSF. For the present study, we compared the DC characteristics exhibited by iDCs and LPS-matured DCs differentiated from PBMCs using GM-CSF and IL-4 or IL-13. Physical characteristics examined include cellular morphology and surface phenotype. Functional traits investigated include FITC-dextran uptake, IL-10 and IL-12 production, allostimulation and cytokine production by stimulated T cells and antigen-specific T cell stimulation. Compared with IL-13-derived DCs, IL-4 treatment yielded more differentiated DCs, with extensive dendrites and higher expression of DC-SIGN, DEC-205, CD86 and HLA-DR. In addition, IL-4 DCs were more efficient at inducing allogeneic T cell proliferation and immature IL-4 DCs had higher endocytic activity at low FITC-dextran concentrations (1 microg ml(-1)). Although IL-13 was capable of generating DCs from PBMCs, it was not as effective as IL-4 in generating DC phenotype and functionality. Thus, the use of GM-CSF and IL-4 is the more efficient treatment for inducing DC differentiation from PBMCs.     

IL-18 produced by thymic epithelial cells induces development of dendritic cells with CD11b in the fetal thymus

Thymic dendritic cells (DCs) are suggested to be involved in T cell selection; however, their exact origin and function remain to be established. Although DCs in the adult thymus are mostly CD8alpha(+)CD11b(-), we found that CD8alpha(-)CD11b(+) DCs were abundantly present in the fetal thymus and they possessed antigen-presenting activity. Interestingly, these CD11b(+) DCs were significantly decreased in mice deficient for TNFR-associated factor 6 (TRAF6), a key signaling molecule downstream of IL-1 and tumor necrosis factor-alpha that have been known to induce DCs from intra-thymic precursor cells. CD11b(+) DCs were induced from CD4(-)CD8(-) thymocytes by fetal thymic epithelial cells (TECs). Analysis of cytokine expression in TECs revealed that none of the cytokines previously shown to induce DCs were expressed. Instead, we found strong expression of IL-18 that transmits signals through TRAF6. IL-18 induced CD11b(+) DCs from CD4(-)CD8(-) thymocytes in vitro, which exhibited strong antigen-presenting activity and formed conjugates with CD4(+)CD8(+) T cells efficiently. Taken together, these results strongly suggest that CD11b(+) DCs are differentiated from CD4(-)CD8(-) thymocytes by IL-18 produced from TECs and that they are involved in T cell selection in the fetal thymus.     

Generation of CD4 and CD8 T Lymphocyte Responses by Dendritic Cells Armed with PSA/Anti-PSA (Antigen/Antibody) Complexes

Dendritic cells (DC) acquire antigens through a number of cell surface structures including receptors for the Fc portion of immunoglobulins and mannose. Little is known about the effects of antigen uptake via these receptors on antigen processing and presentation. We compared the capacity of DC to generate CD4(+) and CD8(+) T cell responses after exposure to prostate-specific antigen (PSA) alone, PSA targeted to the mannose receptor (mannosylated PSA (PSA-m)), or PSA targeted to Fc receptors by combining PSA with an anti-PSA antibody (AR47.47). Autologous CD3(+) T cells were added to monocyte-derived immature DC that had been cultured with GM-CSF/IL-4 for 4 days, exposed to antigen, and matured with CD40L or TNFalpha/IFN-alpha. After several rounds of stimulation, T cell responses were assessed by intracellular IFN-gamma production using flow cytometry. Both CD4(+) and CD8(+) T cell responses were observed after stimulation with DC exposed to the PSA/anti-PSA complexes, whereas CD4(+) predominated over CD8(+) T cell responses after stimulation with PSA-armed DC or PSA-m. These CD8(+) T cells responded when rechallenged with DC pulsed with HLA allele-restricted PSA peptides. These results indicate that PSA and PSA-m are processed primarily through pathways that favor HLA Class II presentation, while the PSA/anti-PSA immune complexes are processed through both Class I and Class II pathways in monocyte-derived DC. These findings have potential applications in designing more effective cancer vaccines for prostate cancer.     

Activation of purified allogeneic CD4(+) T cells by rat bone marrow-derived dendritic cells induces concurrent secretion of IFN-gamma, IL-4, and IL-10

Dendritic cells (DCs) are highly specialized antigen-presenting cells that play a key role in the initiation and regulation of immune responses. The ability of DCs to process antigens and the outcome of their interaction with T cells are largely dependent on phenotype as well as maturation state of DCs. In this study, we generated DCs from rat bone marrow precursors. Bone marrow cells cultured in the presence of granulocyte macrophage colony-stimulating factor (GM-CSF), interleukin (IL)-4, and Flt-3 ligand (FL) produced immature DCs that expressed intermediate levels of major histocompatibility complex (MHC) class II, low levels of CD80 and CD86 molecules and displayed a high capacity of endocytosis. Bone marrow-derived DCs (BMDCs) matured in the presence of lipopolysaccharide (LPS) upregulated expression of MHC class II, CD80 and CD86, while their phagocytic capacity was dramatically reduced. Mature BMDCs stimulated vigorous proliferation of purified allogeneic CD4(+) T cells in a primary mixed leukocyte reaction (MLR) and elicited a mixed cytokine profile in allogeneic CD4(+) T cells: DCs activated CD4(+) T cells to produce interferon (IFN)-gamma, IL-4, and IL-10. Thus, rat BMDCs effectively internalize antigens and stimulate T cell proliferation but fail to induce an unidirectional polarization of T helper (T(H)) cells and in this respect differ from both human and mouse DCs.     

Understanding the immunological basis of asthma; immunotherapy and regulatory T cells

We believe that immunotherapy with HKL as an adjuvant induces Thl-like TReg cells that can inhibit AHR and airway inflammation. These antigen-specific TReg cells are induced with CD8alpha+ DCs producing IL-12 and IL-10, produce IFN-gamma and IL-10, and also express T-bet and Foxp3. These Th1Reg cells are distinct from antigen-specific TReg cells induced with respiratory tolerance, which can also inhibit AHR and airway inflammation. These Th2-like TReg cells are induced with CD8alpha- DCs producing IL-10, they express IL-10, GATA3 and Foxp3. So allergen immunotherapy is effective in large part because it induces regulatory T cells. With HKL you get Th1Reg cells, and with other forms of immunotherapy you may get Th2Reg cells. We believe that with further refinements, allergen immunotherapy that rapidly induces allergen specific TReg cells will indeed be the magic bullets for allergy and asthma.     

Effective induction of acquired resistance to Listeria monocytogenes by immunizing mice with in vivo-infected dendritic cells

Splenic dendritic cells (DCs) obtained from mice at 48 h after Listeria monocytogenes infection exhibited up-regulation of CD80 and produced higher titers of gamma interferon (IFN-gamma) and interleukin-12 (IL-12) than did DCs obtained from uninfected mice. Mice immunized with DCs obtained from mice that had been infected with L. monocytogenes 48 h before acquired host resistance to lethal infection with L. monocytogenes at 4 and 8 weeks. Immunization with DCs from heat-killed L. monocytogenes failed to induce resistance. Acquired antilisterial resistance is specific, since the immunized mice could not be protected from Salmonella enterica serovar Typhimurium infection. Infected DCs stimulated proliferation of naive CD4(+) and CD8(+) cells in vitro, suggesting that in vivo-infected DCs activate CD8(+) T cells, which are critical in acquired antilisterial resistance, as well as CD4(+) T cells. When wild-type mice were immunized with DCs from IFN-gamma-deficient mice, they were protected against a lethal L. monocytogenes challenge. In contrast, when mice were immunized with DCs from anti-IL-12 p40 monoclonal antibody-injected mice, they failed to gain acquired antilisterial resistance. These results suggest that DC-derived IL-12, but not IFN-gamma, may play a critical role in induction of acquired antilisterial resistance. Our present results suggest that splenic DCs obtained from mice infected with L. monocytogenes in vivo may be an effective immunogen with which to induce antigen-specific immunity.     

Comparison of allergen-stimulated dendritic cells from atopic and nonatopic donors dissecting their effect on autologous naive and memory T helper cells of such donors

BACKGROUND: Because of their production of IL-12, mature dendritic cells (DC) are potent inducers of T(H)1 responses. However, recent reports have demonstrated that DCs can also induce T(H)2 differentiation. OBJECTIVE: In the current study we investigated which immune response is induced by DCs in naive CD45RA(+) or memory CD45R0(+) CD4(+) T cells from atopic individuals (patients with grass pollen, birch pollen, or house dust mite allergy) compared with nonatopic control subjects. METHODS: Immature DCs, generated from peripheral blood monocytes from atopic and nonatopic donors, were pulsed with the respective allergen and fully matured. Then the mature DCs were cocultured in vitro with autologous naive (CD45RA(+)) and memory (CD45R0(+)) CD4(+) T cells and cytokine and IgE production were measured by ELISA. RESULTS: After the second restimulation with allergen-pulsed DCs, naive as well as memory autologous CD4(+) T cells from atopic but not from nonatopic donors showed an enhanced production of the T(H)2-type cytokines IL-4, IL-5, and IL-10, resulting in an increased IgE production, whereas IFN-gamma production and proliferation were not different. IL-12 production and surface marker expression of DCs derived from atopic and nonatopic donors did not differ and addition of neutralizing anti-IL-12 mAbs did not increase IL-4 but diminished IFN-gamma production. CONCLUSION: These data indicate that mature DCs are able to induce naive and activate allergen-specific T helper cells to produce T(H)2 cytokines if the T cells are derived from atopic donors. This phenomenon is not due to diminished IL-12 production by DCs of atopic donors.     

Murine keratocytes function as antigen-presenting cells.

Keratocytes express MHC class I molecules constitutively, and keratocytes stimulated with IFN-gamma express MHC class II molecules. Unstimulated keratocytes constitutively express B7-1 and ICAM-1, as well as low levels of CD40 and 4-1BBL. These findings indicate that keratocytes may deliver both antigen-specific and costimulatory signals to CD4(+) and CD8(+) T cells. To demonstrate that keratocytes expressing B7-1 provide a costimulatory signal to T cells, CD4(+) or CD8(+) mouse T cells were incubated with anti-CD3 mAb and irradiated keratocytes. Enhanced proliferation of both CD4(+) and CD8(+) T cells occurred, and could be inhibited by anti-B7-1 mAb, indicating T cell costimulatory activity by B7-1 on the keratocytes. To demonstrate that keratocytes can deliver an antigen-specific signal, CD4(+) and CD8(+) T cells from herpes-infected mice were incubated with HSV-1-infected, irradiated keratocytes. The resulting T cell proliferation and production of Th1 cytokines (IL-2, IFN-gamma) indicated T cell activation by antigens presented by the infected keratocytes. These results show that keratocytes in the corneal stroma of the mouse can function as antigen-presenting cells and, thus, may play a role in immune-mediated stromal inflammation such as herpetic stromal keratitis.     

Maturation of dendritic cells for enhanced activation of anti-HIV-1 CD8(+) T cell immunity

Maturation of dendritic cells (DC) to enhance their capacity to activate T cell immunity to HIV-1 is a key step in immunotherapy of HIV-1 infection with DC. We compared maturation of DC derived from HIV-1-uninfected subjects and infected subjects on antiretroviral therapy (ART) or ART na?ve by CD40 ligand (CD40L) and combinations of TLR3 ligand polyinosinic:polycytidylic acid [poly(I:C)] and inflammatory cytokines IFN-gamma, IFN-alpha, IL-1beta, and TNF-alpha. The greatest levels of virus-specific IFN-gamma production by CD8(+) T cells were stimulated by DC treated with CD40L, followed by DC treated with the poly(I:C)-cytokine combination. The highest levels of IL-12p70 were produced by DC treated with CD40L + IFN-gamma, followed by CD40L and the poly(I:C)-cytokine combination. Neutralization of IL-12p70 indicated that it was only partially involved in direct enhancement of antiviral CD8(+) T cell activity. DC stimulation of antiviral CD8(+) T cell reactivity was enhanced by activated CD4(+) T cells at low concentrations but was suppressed at higher CD4(+) T cell concentrations. Maturation of DC with CD40L obviated the need for CD4(+) T cell help and overcame this suppressive activity. Finally, we showed that DC from HIV-1-infected subjects on ART, which were treated with the poly(I:C)-cytokine combination, retained the capacity to produce IL-12p70 and activate anti-HIV-1 CD8(+) T cell responses after restimulation with CD40L, with or without IFN-gamma. Thus, DC from HIV-1-infected subjects can be engineered with CD40L or a poly(I:C)-cytokine combination for enhancing CD8(+) T cell responses to HIV-1, which has potential applications in HIV-1 immunotherapy.