CD8+ immunoregulatory cells in the graft-versus-host reaction: CD8 T cells activate dendritic cells to secrete interleukin-12/interleukin-18 and induce T helper 1 autoantibody

Initiation of cell-mediated immunity or autoimmunity requires secretion of interleukin (IL)-12 from dendritic cells (DC), which drives the generation of T helper 1 (Th1) effector cells in synergy with IL-18. Induction of IL-12 can be triggered by microbial stimuli but also requires signals from activated T cells. We investigated interactions between alloreactive CD4 and CD8 T cells in mixed lymphocyte reactions (MLR) in vitro and in the graft-versus-host reaction (GVHR) in vivo. In a parent-into-F1 model of GVHR, donor CD8 cells were found to suppress the hyper-immunoglobulin E (IgE) syndrome, anti-DNA immunoglobulin G1 (IgG1) autoantibodies and donor CD4-cell expansion, but were essential for Th1-dependent immunoglobulin G2a (IgG2a) autoantibody production and release of serum IL-12 p40. In vitro, addition of alloreactive CD8 cells to CD4 cells and mature DC enhanced Th1 development. CD4 and CD8 T cells induced IL-18 from DC and primed for IL-12 p70 secretion via interferon-gamma (IFN-gamma) or tumour necrosis factor-alpha (TNF-alpha). However CD8 T cells, but not CD4 cells, released IFN-gamma/TNF-alpha after primary stimulation. The data suggest that rapid release of inflammatory cytokines from central memory-type CD8 cells early in immunity is critical for induction of Th1 cells via DC activation and IL-12 production. This pathway could provide a means for amplification of cell-mediated autoimmunity in the absence of microbial stimuli.     

CD16+ human monocyte-derived dendritic cells matured with different and unrelated stimuli promote similar allogeneic Th2 responses: regulation by pro- and anti-inflammatory cytokines

We previously demonstrated that tumor necrosis factor (TNF)-alpha-matured CD16- and CD16+ human monocyte-derived dendritic cells (16-mDC and 16+mDC) differentially stimulate naive CD4+ lymphocytes by inducing Th1- and Th2-like responses, respectively. Here, we further characterized the role of different DC maturation factors on Th polarization. Immature 16+mDC and 16-mDC (iDC) obtained by culture of purified monocytes with GM-CSF and IL-4 were maturated with (i) Toll-like receptor (TLR) ligands [lipopolysaccharide (LPS)], (ii) lymphocyte-derived (soluble CD40 ligand, IFN-gamma) and (iii) endogenous inflammatory stimuli [TNF-alpha, prostaglandin (PG)E2]. After activation with these stimuli, DC secrete IL-12 only in presence of LPS, and 16+mDC produced lower amounts of IL-12 and IL-10 than 16-mDC. Allogeneic CD4+CD45RO- lymphocytes co-cultured with 16+mDC secreted higher levels of IL-4 and IL-10 than those co-cultured with 16-mDC, regardless of the maturation stimuli. Results were similar when DC were activated with TLR-2 or TLR-3 ligands. The higher induction of IL-4 by 16+mDC was primarily dependent on IL-12, IL-4 and IL-10. IFN-gamma production by CD4+ T cells was similar with all the conditions except with LPS-16+mDC, which induced reduced amounts of this cytokine. Those differences were totally eliminated by neutralization of IL-12, IL-4 or IL-10. Finally, 16-mDC could reverse the Th2 phenotype of already committed lymphocytes toward a Th1 pattern in short-term cultures, whereas 16+mDC had less ability to skew this phenotype. These results indicate that 16+mDC elicit superior Th2 responses independently of the maturation factors that they received, and suggest that they could represent an important population of regulatory DC.     

OX40-Mediated cosignal enhances the maturation of naive human CD4+ T cells into high IL-4-producing effectors

Our previous studies have indicated that naive human CD4+ T cells of neonatal or adult origin may be the initial source of IL-4 which is required for their development into Th2 effectors. In addition to minute amounts of IL-4, anti-CD3/B7.1-activated naive cells also release readily detectable levels of IL-13 and IFN-gamma. The production of IL-4 and IL-13 by naive T cells is differentially regulated by TGF-beta and IL-12. Shortly after activation, naive T cells express surface OX40, a TNF-R family member whose ligand (OX40L) is constitutively expressed on a subset of dendritic cells. Engagement of OX40 on activated naive T cells increases their expression of IL-4 and IL-13, suppresses that of IFN-gamma and promotes their development into Th2-like effectors.     

IFN-alpha enhances CD40 ligand-mediated activation of immature monocyte-derived dendritic cells

Type I IFN are immune modulatory cytokines that are secreted during early stages of infection. Type I IFN bridge the innate and the adaptive immune system in humans and mice. We compared the capacity of type I and II IFN to induce the functional maturation of monocyte-derived dendritic cells (MoDC). Extending our earlier observation that type I IFN promote DC maturation, we report that these cytokines also enhance DC differentiation by augmenting CD40 ligand (CD40L)-induced cytokine secretion by MoDC. Type I IFN alone were poor inducers of MoDC maturation as compared with other stimuli. They up-regulated the expression of HLA-DR, CD80, CD86, partially CCR7 but not CD83, partially reduced antigen-uptake function, increased the levels of IL-12p35 mRNA, and prolonged surface expression of peptide-MHC class I complexes for presentation to cytotoxic T lymphocytes, but did not induce migration towards CCL21 chemokine. However, type I IFN were potent co-factors for CD40L-mediated function. Here, they enhanced CD40L-mediated IL-6, IL-10 and IL-12p70 secretion. Furthermore, when combined with IL-1beta and/or IL-4, IFN-alpha2a type I IFN increased CD40L-mediated IL-12p70 production by 2- to 3-fold, and biased the IL-12 p40/p70 ratio towards the IFN-gamma inducing p70 heterodimer, this correlating with higher levels of IFN-gamma secretion by allogeneic T cell subsets and NK cells. Our results suggest that the rapid expression of CD40L, IFN and IL-1beta at sites of infection and inflammation can act in concert on immature DC, thereby linking innate and adaptive immune responses. In this way, type I IFN play a dual role as DC maturation factors and enhancers of CD40L-mediated DC activation.     

Dendritic cells differentiated in the presence of IFN-{beta} and IL-3 are potent inducers of an antigen-specific CD8+ T cell response

Dendritic cells (DC) are professional antigen-presenting cells that are used in vaccine approaches to cancer. Classically, mature monocyte-derived DC are generated in vitro in the presence of interleukin (IL)-4, granulocyte macrophage-colony stimulating factor, and inflammatory cytokines (G4-DC). Recently, it has been described that DC can also be generated in the presence of IL-3 and interferon (IFN)-beta and that these DC are efficiently matured using polyriboinosinic polyribocytidylic acid (I3-DC). In this study, a series of in vitro experiments was performed to compare side-by-side I3-DC and G4-DC as vaccine adjuvants. Phenotypic characterization of the DC revealed differences in the expression of the monocyte marker CD14 and the maturation marker CD83. Low expression of CD14 and high expression of CD83 characterized G4-DC, whereas I3-DC displayed intermediate expression of CD14 and CD83. Both types of DC were as potent in the induction of allogeneic T cell proliferation. Upon CD40 ligation, G4-DC produced lower amounts of IFN-alpha and pulmonary and activation-regulated chemokine, similar amounts of IL-6, macrophage-inflammatory protein (MIP)-1alpha, and MIP-1beta, and higher amounts of IL-12 p70, tumor necrosis factor alpha, and MIP-3beta than I3-DC. We further evaluated whether the DC could be frozen/thawed without loss of cell number, viability, phenotype, and function. After freezing/thawing, 56.0% +/- 9.0% of I3-DC and 77.0% +/- 3.0% of G4-DC (n=9) were recovered as viable cells, displaying the same phenotype as their fresh counterparts. Finally, in vitro stimulations showed that fresh and frozen peptide-loaded I3-DC are more potent inducers of Melan-A-specific CD8(+) T cell responses than G4-DC. The antigen-specific T cells were functional as shown in cytotoxicity and IFN-gamma secretion assay.     

Interferon-gamma Added During Bacillus Calmette-Guerin Induced Dendritic Cell Maturation Stimulates Potent Th1 Immune Responses

Dendritic cells (DC) are increasingly prepared in vitro for use in immunotherapy trials. Mature DC express high levels of surface molecules needed for T cell activation and are superior at antigen-presentation than immature DC. Bacillus Calmette-Guerin (BCG) is one of several products known to induce DC maturation, and interferon (IFN)-gamma has been shown to enhance the activity of DC stimulated with certain maturation factors. In this study, we investigated the use of IFN-gamma in combination with the powerful maturation agent, BCG. The treatment of immature DC with IFN-gamma plus BCG led to the upregulation of CD54, CD80, and CD86 in comparison with BCG treatment alone. In MLR or recall immune responses, the addition of IFN-gamma at the time of BCG-treatment did not increase the number of antigen-specific T cells but enhanced the development of IFN-gamma-producing Th1 cells. In primary immune responses, on the other hand, BCG and IFN-gamma co-treated DC stimulated higher proportions of specific T cells as well as IFN-gamma secretion by these T cells. Thus the use of IFN-gamma during BCG-induced DC maturation differentially affects the nature of recall versus na?ve antigen-specific T-cell responses. IFN-gamma co-treatment with BCG was found to induce IL-12 and, in some instances, inhibit IL-10 secretion by DC. These findings greatly enhance the potential of BCG-matured dendritic cells for use in cancer immunotherapy.     

Costimulatory effects of IL-1 on the expansion/differentiation of CD4+CD25+Foxp3+ and CD4+CD25+Foxp3- T cells

CD4+CD25+forkhead box p3 (Foxp3)+ regulatory T cells (Treg) control peripheral tolerance. Although Treg are anergic when stimulated through the TCR, mature bone marrow-derived, but not splenic, dendritic cells (DC) can induce their proliferation after TCR stimulation in the absence of IL-2. One possibility is that the DC produce proinflammatory cytokines such as IL-1 or IL-6 that function as growth factors for Treg. We have analyzed the costimulatory effects of IL-1 on the expansion of Foxp3+ Treg in vitro. When CD4+CD25+ T cells were cultured in the presence of splenic DC and IL-1, marked expansion of the Foxp3+ T cells was observed. The effects of IL-1 were mediated on CD4+CD25+Foxp3(-) T cells present in the starting population rather than on the DC or on the CD4+CD25+Foxp3+ T cells. In contrast, stimulation of CD4+CD25+ T cells with plate-bound anti-CD3 and IL-1 in the absence of DC resulted in the outgrowth of a CD4+CD25+Foxp3(-) T cell population composed of NKT cells and non-NKT, IL-17-producing cells. Foxp3+ Treg purified from mice expressing the reporter gene enhanced GFP in the Foxp3 locus failed to proliferate when costimulated with IL-1. These findings have important implications for the design of protocols for the expansion of CD4+CD25+ T cells for cellular biotherapy.     

Human T cells elicit IFN-alpha secretion from dendritic cells following cell to cell interactions

Major insights into events that control Th1/Th2 differentiation have been acquired recently, and highlight the role of Type I IFN in Th1 generation, by inducing up-regulation of the IL-12 receptor beta(2) subunit. IFN-alpha induces responsiveness to IL-12, and here we have investigated the source and the circumstances under which IFN-alpha is produced, in the absence of viral infections. Human dendritic cells (DC) were co-cultured with autologous T cells activated by cross-linking the CD3 complex. DC were also cultured with L cells expressing human CD40 ligand (CD40L). Our results show that large amounts (>200 IU IFN-alpha from 2.5x10(4) cells) of IFN-alpha are produced by DC following interaction with stimulated T cells. Similar effects were observed when DC were cultured with CD40L-expressing transfectants, although the amount of IFN-alpha produced was reduced, suggesting that the CD40-CD40L interaction may be important. These results show that stimulated T cells can solicit the signals from DC that allow their polarization towards a Th1 phenotype. Type I DC produce Type I IFN not only following viral infection but also during an immunological interaction and this may be the basic mechanism that assists in the development of a Th1 response.     

Activation of human NK cells by plasmacytoid dendritic cells and its modulation by CD4+ T helper cells and CD4+ CD25hi T regulatory cells

Plasmacytoid dendritic cells (pDC) represent a specialized cell population that produce type I interferon (IFN) in response to virus. Although type I IFN is a natural killer (NK) cell modulator, a direct role for pDC in coordinating NK cell functions has not yet been elucidated in detail, especially in humans. Here we report that human pDC, following engagement of Toll-like receptor (TLR) 9, not only activate autologous NK cells, as indicated by the induction of CD69 expression, but also enhance their effector functions, especially cytotoxicity. Moreover, they can induce selective proliferation of CD56bright CD16- NK cells. This activity can be strongly augmented by the addition of autologous CD4+ CD25- T helper cells in an IL-2-dependent fashion. Strikingly, CD4+ CD25hi T regulatory (Treg) cells completely abrogate this IL-2-dependent proliferation of NK cells, while they are not able to influence NK cell activation or proliferation solely induced by pDC. Our data show that TLR9-engaged pDC represent a critical stimulus for human NK cells and that CD4+ Th cells and CD4+ CD25hi Treg cells play an important role in modulating human NK cell responses.     

Instruction of naive CD4+ T cells by polarized CD4+ T cells within dendritic cell clusters

Cooperation between CD4(+) T cells can enhance the response and modulate the cytokine profile, and defining these parameters has become a major issue for multivalent-vaccine strategies.We explored cooperation using adoptive transfer of two populations of TCR transgenic T cells of different specificity. One was transferred without prior activation, whereas the second was activated for five days by antigen stimulation under polarizing culture conditions. Both populations were transferred into a single adoptive host and then primed by particle-mediated DNA delivery. Polarized Th1 cells (inducers) raised the frequency of IFN-gamma(+) cells within a naive (target) population, whereas Th2 inducers raised the frequency of IL-4(+) and reduced that of IL-2(+) cells. These effects were obtained when the genes for both antigens were on the same particle, favoring presentation by the same dendritic cell, but not when on different particles delivered to different dendritic cells. Autonomy of DC clusters allows linked sets of antigens (e.g. from a single pathogen) to maintain cytokine bias, but allows other independent responses, each with their own set of autonomous clusters.     

Combined cyclosporin-A /prednisone therapy of patients with active uveitis suppresses IFN-gamma production and the function of dendritic cells

In this study, we assessed the Th1/Th2 polarization of the immune response and the involvement of dendritic cells (DC) and Th1 lymphocytes in the pathogenesis of uveitis. Thirty-seven patients with chronic idiopathic uveitis were enrolled: 21 of them had active uveitis and the remaining 16 were in complete remission. Patients with active uveitis were characterized as follows: 5 had intermediate uveitis, 5 panuveitis and the remaining 11 posterior uveitis. Thirteen healthy subjects were also studied as controls. Patients with active uveitis were treated with cyclosporin-A (CsA) associated to low doses of prednisone (PDS) and studied at baseline and after 6 months of therapy. Analysis of cytokine-producing CD3+ lymphocytes revealed a strong Th1 polarization of the immune response in patients with active uveitis. Th1 lymphocytes paralleled serum IL-12 levels and the response to therapy, which greatly reduced both IFN-gamma+/CD3+ lymphocytes and serum IL-12 levels, associated with a general clinical improvement. In vitro studies demonstrated that DC from untreated patients with active uveitis were mature and functionally active. In fact, they showed a higher ability to stimulate cell proliferation of allogeneic T cells in primary mixed lymphocyte reaction (MLR) and produced larger amounts of IL-12 than DC from CsA/PDS-treated patients and those in remission. These results demonstrate that CsA/PDS therapy impairs the capacity of mature DC to secrete IL-12 and inhibits their MLR activity.     

Interferon-gamma is an autocrine mediator for dendritic cell maturation

Maturation of dendritic cells (DC) is critical for efficient antigen presentation and initiation of an immune response. Interferon-gamma (IFN-gamma) is an important Th1 cytokine. In this study, we investigated the role of IFN-gamma in DC maturation using either IFN-gamma receptor deficient- or IFN-gamma overexpression-models. We showed that immature DC generated in vitro from bone marrow (BM) progenitor cells produced low level of IFN-gamma. After LPS stimulation, DC produced more IFN-gamma, and IFN-gamma productions were at comparable levels among C57BL/6 mice-derived DC (C57BL/6 DC), wild-type 129 mice-derived DC (129 DC) and IFN-gamma receptor deficient 129 mice-derived DC (IFN-gammaR-/-DC). We found that IFN-gammaR-/-DC exhibited decreased expression of CD54, CD86, reduced capacity to secrete IL-1beta and IL-12p70, and impaired capacity to stimulate alloreactive T cells and to drive Th1 differentiation. Transfection of IFN-gamma gene into DC promoted DC to express higher CD40, CD54, CD80, CD86, CCR7 and I-Ab, secrete more IL-1beta and IL-12p70, and more potently activate both CD4 and CD8 T cells. These data suggest that IFN-gamma signaling pathway is important for the maturation of DC in an autocrine fashion.     

Increased numbers of Th2-like CD4+ T cells in target organs and in the allergen-specific repertoire of allergic patients. Possible role of IL-4 produced by non-T cells.

Phytohemagglutinin (PHA)-induced human T cell clones (TCC) derived from conjunctival flogistic tissues of 3 patients with vernal conjunctivitis produced unusually high amounts of interleukin-4 (IL-4) and no, or limited amounts of, gamma-interferon (IFN-gamma). Allergen (Dermatophagoides pteronyssinus or Lolium perenne group I)-specific TCC derived from peripheral blood of two atopic donors produced significantly higher amounts of IL-4 and significantly lower amounts of IFN-gamma than TCC specific for bacterial antigens (tetanus toxoid and PPD) contemporarily established from the same donors. These data provide evidence for a compartimentalization of Th2-like helper T cells in target organs and in the allergen-specific T cell repertoire of allergic patients. Non-B, non-T bone marrow cells could produce IL-4, but not IL-2 or IFN-gamma, in response to cross-linkage of Fc epsilon type I receptors. These cells may further contribute to the maintenance and amplification of allergic inflammation.     

The phenotype of type 1 and type 2 CD8+ T cells activated in vitro is affected by culture conditions and correlates with effector activity

We used various culture conditions to generate type 1 (Tc1) or type 2 (Tc2) cytotoxic T cells in vitro. T-cell receptor (TCR) transgenic T cells were cultured with antigen and spleen cells, or antigen and dendritic cells (DC), or anti-CD3 and anti-CD28. Tc1 cultures contained interleukin (IL)-2 and IL-6, and Tc2 cultures contained IL-2, IL-6 and IL-4. Tc2 cells generated in each culture condition acquired a CD62L(low) CD44(high) phenotype, had high cytotoxic activity, and secreted IL-4, IL-5 and moderate amounts of interferon-gamma (IFN-gamma). In contrast, the phenotype and function of Tc1 cells varied depending on culture conditions. Tc1 cells from anti-CD3 and anti-CD28 cultures had high cytotoxic activity and were CD62L(low) CD44(high), while Tc1 cells from antigen and spleen cell cultures had low cytotoxic activity and were CD62L(high) CD44(low). Tc1 cells from antigen and DC cultures had an intermediate phenotype. All Tc1 cells secreted high amounts of IFN-gamma, but only Tc1 from anti-CD3 and anti-CD28 cultures had antitumour activity in vivo. Differences were not caused by suboptimal culture conditions, as Tc1 cells divided at a similar rate whether cultured with antigen and spleen cells or with anti-CD3 and anti-CD28. We conclude that IL-4 not only induces 'type 2' cytokine secretion in CD8(+) T cells, but also affects their expression of surface markers and cytotoxic activity.     

Hepatitis C virus non-structural protein 4 suppresses Th1 responses by stimulating IL-10 production from monocytes

The majority of hepatitis C virus (HCV) infections become chronic, despite the presence of HCV-specific cellular and humoral immune responses. We have previously suggested that IL-10-secreting antigen-specific regulatory T cells may contribute to viral persistence, and demonstrate here that peripheral blood mononuclear cells (PBMC) from chronically HCV-infected patients secrete IL-10, but not IFN-gamma, in response to HCV nonstructural protein 4 (NS4). A neutralizing anti-IL-10 antibody restored this defective antigen-specific IFN-gamma production in vitro. Furthermore, PBMC from normal individuals secreted IL-10 in response to NS4, suggesting that cells of the innate immune system, in addition to T cells, produced IL-10 in the HCV-infected patients. Cell separation experiments revealed that the innate IL-10 was produced by blood monocytes, but not dendritic cells (DC). In addition, NS4 inhibited IL-12 production by PBMC in response to LPS and IFN-gamma, and Th1 responses to recall antigens in normal individuals. Furthermore, supernatants from NS4-stimulated monocytes inhibited LPS-induced maturation of DC and suppressed their capacity to stimulate proliferation and IFN-gamma production by allospecific T cells. Our data suggest that HCV subverts cellular immunity by inducing IL-10 and inhibiting IL-12 production by monocytes, which in turn inhibits the activation of DC that drive the differentiation of Th1 cells.     

Differential regulation of Th1 responses and CD154 expression in human CD4+ T cells by IFN-alpha

Like interleukin (IL)-12, interferon (IFN)-alpha has been shown to play an important role in inducing human Th1 responses. Recent studies have shown that human Th1 responses driven by IL-12 are associated with enhanced expression of CD154. The present study examined the effects of IFN-alpha on CD154 expression in human CD4+ T cells, with special attention to the relationship with Th1 responses. Highly purified CD4+ T cells from healthy donors were stimulated with immobilized anti-CD3 with or without IFN-alpha and IL-12 in the complete absence of accessory cells. IFN-alpha suppressed CD154 protein and mRNA expression in CD4+ T cells at the initial phase of activation with immobilized anti-CD3, but enhanced it in the subsequent maturation phase irrespective of the presence of IL-12. By contrast, IFN-alpha by itself did not enhance IFN-gamma production or mRNA expression in CD4+ T cells in the absence of IL-12 even in the presence of stimulation with anti-CD28, but enhanced it in the presence of IL-12. Accordingly, IFN-alpha enhanced IL-12Rbeta2 mRNA expression in anti-CD3-stimulated CD4+ T cells. Neither IFN-alpha nor IL-12 influenced the stability of CD154 mRNA in anti-CD3-activated CD4+ T cells. These results indicate that IFN-alpha by itself enhances CD154 expression in CD4+ T cells independently of the induction of IFN-gamma mRNA expression. The data also suggest that the optimal induction of human Th1 responses by IFN-alpha might require the presence of IL-12 and that the induction of Th1 responses and CD154 expression in human CD4+ T cells might be regulated through different mechanisms.     

Induction of human CD4+ regulatory T cells by mycophenolic acid-treated dendritic cells

Depending on their degree of maturation, costimulatory molecule expression, and cytokine secretion, dendritic cells (DC) can induce immunity or tolerance. DC treated with mycophenolic acid during their maturation (MPA-DC) have a regulatory phenotype and may therefore provide a new approach to induce allograft tolerance. Purified CD4(+) T cells stimulated in a human in vitro model of mixed culture by allogeneic MPA-DC displayed much weaker proliferation than T cells activated by mature DC and were anergic. This hyporesponsiveness was alloantigen-specific. Interestingly, T cells stimulated by MPA-DC during long-term coculture in four 7-day cycles displayed potent, suppressive activity, as revealed by marked inhibition of the proliferation of naive and preactivated control T cells. These regulatory T cells (Tregs) appeared to have antigen specificity and were contact-dependent. Tregs induced by MPA-DC were CD25(+)glucocorticoid-induced TNFR(+)CTLA-4(+)CD95(+), secreted IL-5 and large amounts of IL-10 and TGF-beta, and displayed enhanced forkhead box p3 expression. These results obtained in vitro demonstrate that human MPA-DC can induce allospecific Tregs that may be exploited in cell therapy to induce allograft tolerance.