Immature dendritic cells acquire CD8(+) cytotoxic T lymphocyte priming capacity upon activation by T helper cell-independent or -dependent stimuli

The well defined, immature murine dendritic cell (DC) line D1 was used to study the role of DC maturation in CTL induction in vitro and in vivo. Maturation of D1 cells, characterized by markedly increased expression of MHC and costimulatory molecules, was induced by incubation with lipopolysaccharide, agonistic CD40 antibody, or specific CD4(+) T helper (Th) cells. Activated, but not immature, D1 cells efficiently primed alloreactive T cell responses in vitro. Similarly, priming of CTL immunity in vivo in CD4-depleted mice was only observed if these mice were immunized with activated D1 cells. This study provides formal evidence that activation of DCs, induced by Th-independent as well as Th-dependent stimuli, is essential for efficient induction of CTL responses.     

CD40-independent pathways of T cell help for priming of CD8(+) cytotoxic T lymphocytes

In many cases, induction of CD8(+) CTL responses requires CD4(+) T cell help. Recently, it has been shown that a dominant pathway of CD4(+) help is via antigen-presenting cell (APC) activation through engagement of CD40 by CD40 ligand on CD4(+) T cells. To further study this three cell interaction, we established an in vitro system using dendritic cells (DCs) as APCs and influenza hemagglutinin (HA) class I and II peptide-specific T cell antigen receptor transgenic T cells as cytotoxic T lymphocyte precursors and CD4(+) T helper cells, respectively. We found that CD4(+) T cells can provide potent help for DCs to activate CD8(+) T cells when antigen is provided in the form of either cell lysate, recombinant protein, or synthetic peptides. Surprisingly, this help is completely independent of CD40. Moreover, CD40-independent CD4(+) help can be documented in vivo. Finally, we show that CD40-independent T cell help is delivered through both sensitization of DCs and direct CD4(+)-CD8(+) T cell communication via lymphokines. Therefore, we conclude that CD4(+) help comprises at least three components: CD40-dependent DC sensitization, CD40-independent DC sensitization, and direct lymphokine-dependent CD4(+)-CD8(+) T cell communication.     

A subset of dendritic cells induces CD4+ T cells to produce IFN-gamma by an IL-12-independent but CD70-dependent mechanism in vivo

Interferon (IFN)-gamma, a cytokine critical for resistance to infection and tumors, is produced by CD4(+) helper T lymphocytes after stimulation by cultured dendritic cells (DCs) that secrete a cofactor, interleukin (IL)-12. We have identified a major IL-12-independent pathway whereby DCs induce IFN-gamma-secreting T helper (Th)1 CD4(+) T cells in vivo. This pathway requires the membrane-associated tumor necrosis family member CD70 and was identified by targeting the LACK antigen from Leishmania major within an antibody to CD205 (DEC-205), an uptake receptor on a subset of DCs. Another major DC subset, targeted with 33D1 anti-DCIR2 antibody, also induced IFN-gamma in vivo but required IL-12, not CD70. Isolated CD205(+) DCs expressed cell surface CD70 when presenting antigen to T cell receptor transgenic T cells, and this distinction was independent of maturation stimuli. CD70 was also essential for CD205(+) DC function in vivo. Detection of the IL-12-independent IFN-gamma pathway was obscured with nontargeted LACK, which was presented by both DC subsets. This in situ analysis points to CD70 as a decision maker for Th1 differentiation by CD205(+) DCs, even in Th2-prone BALB/c animals and potentially in vaccine design. The results indicate that two DC subsets have innate propensities to differentially affect the Th1/Th2 balance in vivo and by distinct mechanisms.     

CD8- DCs induce IL-12-independent Th1 differentiation through Delta 4 Notch-like ligand in response to bacterial LPS

Toll-like receptor (TLR) ligation is believed to skew T cell responses toward T helper (Th)1 differentiation by inducing interleukin (IL)-12 secretion by CD8(+) dendritic cells (DCs). However, TLR-dependent Th1 responses occur in the absence of IL-12. To determine how DCs induce Th1 differentiation in the absence of IL-12, we examined the response of IL-12-deficient DCs to bacterial lipopolysaccharide (LPS). We find that LPS activates MyD88-dependent Delta 4 Notch-like ligand expression by CD8(-) DCs, and that these cells direct Th1 differentiation by an IL-12-independent and Notch-dependent mechanism in vitro and in vivo. Thus, activation of the two DC subsets by TLR4 leads to Th1 responses by two distinct MyD88-dependent pathways.     

Generation of tumor-associated cytotoxic T lymphocytes requires interleukin 4 from CD8(+) T cells.

Activation of tumor-associated CD8(+) cytotoxic T lymphocytes (CTLs) often requires antigen representation, e.g., by dendritic cells (DCs), and CD4(+) T cell help. Previously, we showed that CTL-mediated tumor immunity required interleukin 4 (IL-4) during the immunization but not effector phase. To determine the source and target cells of IL-4, we performed adoptive T cell transfers using CD4(+) and CD8(+) T cells from IL-4(-/-) and IL-4R(-/-) mice and analyzed CTL generation. Even though necessary for CTL generation, CD4(+) T cells did not need to express IL-4 or IL-4R. Surprisingly, CTL generation required IL-4 but not IL-4R expression by CD8(+) T cells. As IL-4 (a) was expressed by naive CD8(+) T cells within 24 h after antigen encounter, (b) IL-4 induced DC maturation, and (c) CTL development was impaired in T cell-reconstituted IL-4R(-/-) mice, CD8(+) T cell-derived IL-4 appears to act on DCs. We conclude that CD4(+) and CD8(+) T cells provide different signals for DC activation during CTL generation.     

CD103+ pulmonary dendritic cells preferentially acquire and present apoptotic cell-associated antigen

Cells undergoing programmed cell death (apoptosis) are removed in situ by macrophages and dendritic cells (DCs) through a specialized form of phagocytosis (efferocytosis). In the lung, there are two primary DC subsets with the potential to migrate to the local lymph nodes (LNs) and initiate adaptive immune responses. In this study, we show that only CD103(+) DCs were able to acquire and transport apoptotic cells to the draining LNs and cross present apoptotic cell-associated antigen to CD8 T cells. In contrast, both the CD11b(hi) and the CD103(+) DCs were able to ingest and traffic latex beads or soluble antigen. CD103(+) DCs selectively exhibited high expression of TLR3, and ligation of this receptor led to enhanced in vivo cytotoxic T cell responses to apoptotic cell-associated antigen. The selective role for CD103(+) DCs was confirmed in Batf3(-/-) mice, which lack this DC subtype. Our findings suggest that CD103(+) DCs are the DC subset in the lung that captures and presents apoptotic cell-associated antigen under homeostatic and inflammatory conditions and raise the possibility for more focused immunological targeting to CD8 T cell responses.     

CD8(+) but not CD8(-) dendritic cells cross-prime cytotoxic T cells in vivo

Bone marrow-derived antigen-presenting cells (APCs) take up cell-associated antigens and present them in the context of major histocompatibility complex (MHC) class I molecules to CD8(+) T cells in a process referred to as cross-priming. Cross-priming is essential for the induction of CD8(+) T cell responses directed towards antigens not expressed in professional APCs. Although in vitro experiments have shown that dendritic cells (DCs) and macrophages are capable of presenting exogenous antigens in association with MHC class I, the cross-presenting cell in vivo has not been identified. We have isolated splenic DCs after in vivo priming with ovalbumin-loaded beta2-microglobulin-deficient splenocytes and show that they indeed present cell-associated antigens in the context of MHC class I molecules. This process is transporter associated with antigen presentation (TAP) dependent, suggesting an endosome to cytosol transport. To determine whether a specific subset of splenic DCs is involved in this cross-presentation, we negatively and positively selected for CD8(-) and CD8(+) DCs. Only the CD8(+), and not the CD8(-), DC subset demonstrates cross-priming ability. FACS((R)) studies after injection of splenocytes loaded with fluorescent beads showed that 1 and 0.6% of the CD8(+) and the CD8(-) DC subsets, respectively, had one or more associated beads. These results indicate that CD8(+) DCs play an important role in the generation of cytotoxic T lymphocyte responses specific for cell-associated antigens.     

CD8alpha- CD11b+ dendritic cells present exogenous virus-like particles to CD8+ T cells and subsequently express CD8alpha and CD205 molecules

Recombinant porcine parvovirus virus-like particles (PPV-VLPs) are particulate exogenous antigens that induce a strong, specific cytotoxic T lymphocyte (CTL) response in the absence of adjuvant. In the present report, we demonstrate in vivo that dendritic cells (DCs) present PPV-VLPs to CD8+ T cells after intracellular processing. PPV-VLPs are captured by DCs with a high efficacy, which results in the delivery of these exogenous antigens to 50% of the whole spleen DC population. In vivo, a few hours after injection, PPV-VLPs are presented exclusively to CD8+ T cells by CD8alpha- DCs, whereas 15 hours later they are presented mainly by CD8alpha+ DCs. After PPV-VLPs processing, a fraction of CD11b+ DCs undergo phenotypic changes, i.e., the up-regulation of CD8alpha and CD205 and the loss of CD4 molecules on their surface. The failure to detect mRNA coding for CD8alpha in CD11b+ DCs suggests that CD8alpha expression by these cells is not due to de novo synthesis. In recombination-activating gene knockout mice (Rag-/-), CD11b+ DCs did not express CD8alpha and PPV-VLPs presentation by CD8alpha+ DCs was severely diminished. These results indicate that both CD8alpha- and CD8alpha+ DCs play an important role in the induction of CTL responses by exogenous antigens, such as VLP.     

The linkage of innate to adaptive immunity via maturing dendritic cells in vivo requires CD40 ligation in addition to antigen presentation and CD80/86 costimulation

Dendritic cell (DC) maturation is an innate response that leads to adaptive immunity to coadministered proteins. To begin to identify underlying mechanisms in intact lymphoid tissues, we studied alpha-galactosylceramide. This glycolipid activates innate Valpha14(+) natural killer T cell (NKT) lymphocytes, which drive DC maturation and T cell responses to ovalbumin antigen. Hours after giving glycolipid i.v., tumor necrosis factor (TNF)-alpha and interferon (IFN)-gamma were released primarily by DCs. These cytokines induced rapid surface remodeling of DCs, including increased CD80/86 costimulatory molecules. Surprisingly, DCs from CD40(-/-) and CD40L(-/-) mice did not elicit CD4(+) and CD8(+) T cell immunity, even though the DCs exhibited presented ovalbumin on major histocompatibility complex class I and II products and expressed high levels of CD80/86. Likewise, an injection of TNF-alpha up-regulated CD80/86 on DCs, but CD40 was required for immunity. CD40 was needed for DC interleukin (IL)-12 production, but IL-12p40(-/-) mice generated normal ovalbumin-specific responses. Therefore, the link between innate and adaptive immunity via splenic DCs and innate NKT cells has several components under distinct controls: antigen presentation in the steady state, increases in costimulatory molecules dependent on inflammatory cytokines, and a distinct CD40/CD40L signal that functions together with antigen presentation ("signal one") and costimulation ("signal two") to generate functioning CD4(+) T helper cell 1 and CD8(+) cytolytic T lymphocytes.     

Potent maturation of monocyte-derived dendritic cells after CD40L lentiviral gene delivery

Dendritic cells (DCs) are being evaluated in immunization protocols to enhance immunity against infectious diseases and cancer. Interaction of T-helper cells expressing CD40 ligand (CD40L) with its cognate CD40 receptor on DCs leads to a mature DC phenotype, characterized by increased capacity of antigen presentation to cytotoxic T cells. The authors examined the ability of third-generation self-inactivating lentiviral vectors expressing CD40L to induce autonomous maturation of ex vivo expanded human monocyte-derived dendritic cells. Transduction with lentiviral vectors achieved a highly efficient gene transfer of CD40L to DCs, which correlated with phenotypic maturation as shown by the expression of immunologic relevant markers (CD83, CD80, MHCI) and secretion of IL-12, whereas DC phenotype was not affected by a control vector expressing only the green fluorescent protein marker. Addition of recombinant IFN-gamma to DCs at the time of CD40L transduction further enhanced IL-12 production, and when co-cultured with allogeneic and autologous CD8+ and CD4+ T cells, a potent activation was observed. Autologous responses against an HLA-A2-restricted influenza peptide (Flu-M1) and a tumor-associated antigenic peptide (gp100 210M) were significantly enhanced when CD40L transduced DCs were used as antigen-presenting cells for in vitro stimulation of CD8+ cytotoxic T lymphocytes. These results demonstrate that endogenous expression of CD40L by lentivirally transduced DCs induced their autonomous maturation to a phenotype comparable to that induced by optimal concentrations of soluble CD40L, providing a novel tool for genetic manipulation of DCs.     

CD4+ T cells are required for development of a murine retrovirus- induced immunodeficiency syndrome (MAIDS)

Mice depleted in vivo of CD4+ Th cells by treatment with mAb GK1.5 were found to be resistant to the lymphoproliferative/immunodeficiency disease (MAIDS) induced in intact mice by infection with the mixture of LP-BM5 murine leukemia viruses. Depleted mice did not develop lymphadenopathy or splenomegaly, had normal serum IgM levels, normal CTL responses to alloantigens, and were able to generate PFC responses to Th-independent antigens even though frequencies of virus-producing spleen cells were comparable in depleted and intact mice. Depletion of CD4+ Th cells after infection resulted in a reversal of many abnormalities exhibited by infected controls; spleen weights, serum IgM levels, and allogeneic CTL responses of treated mice were comparable to those of uninfected controls. These results demonstrate that dysfunction of CD4+ Th cells is central to the induction and progression of both T and B cell abnormalities in MAIDS.     

Natural killer-dendritic cell cross-talk in cancer immunotherapy

Natural killer (NK) cells and dendritic cells (DCs), two important components of the immune system, can exchange bidirectional activating signals in a positive feedback. Myeloid DCs, the cell type specialised in the presentation of antigen and initiation of antigen-specific immune responses, have recently been documented to be involved in supporting innate immunity, promoting the production of cytokines and cytotoxicity of NK cells, and enhancing their tumouricidal activity. Natural interferon-producing cells/plasmacytoid DCs (IPCs/PDCs) play an additional role in NK cell activation. Reciprocally, NK cells, traditionally considered to be major innate effector cells, have also recently been shown to play immunoregulatory 'helper' functions, being able to activate DCs and to enhance their ability to produce pro-inflammatory cytokines, and to stimulate T helper (Th) 1 and cytotoxic T lymphocyte (CTL) responses of tumour-specific CD4+ and CD8+ T cells. Activated NK cells induce the maturation of myeloid DCs into stable type-1 polarised DCs (DC1), characterised by up to a 100-fold enhanced ability to produce IL-12p70 in response to subsequent interaction with Th cells. In addition, the ability of NK cells to kill tumour cells may facilitate the generation of tumour-related antigenic material, further accelerating the induction of tumour-specific immunity. DC1, induced by NK cells or by NK cell-related soluble factors, are stable, resistant to tumour-related suppressive factors, and demonstrate a strongly enhanced ability to induce Th1 and CTL responses in human in vitro and mouse in vivo models. Compared with the standard mature DCs that are used in clinical trials at present, human NK cell-induced DC1s act as superior inducers of anticancer CTL responses during in vitro sensitisation. This provides a strong rationale for the combined use of NK cells and DCs in the immunotherapy of patients with cancer and patients with chronic infections that are resistant to standard forms of treatment. Stage I/II clinical trials that are being implemented at present should allow evaluation of the immunological and clinical efficacy of combined NK-DC therapy of melanoma and other cancers.     

Dyslipidemia inhibits Toll-like receptor-induced activation of CD8alpha-negative dendritic cells and protective Th1 type immunity

Environmental factors, including diet, play a central role in influencing the balance of normal immune homeostasis; however, many of the cellular mechanisms maintaining this balance remain to be elucidated. Using mouse models of genetic and high-fat/cholesterol diet-induced dyslipidemia, we examined the influence of dyslipidemia on T cell and dendritic cell (DC) responses in vivo and in vitro. We show that dyslipidemia inhibited Toll-like receptor (TLR)-induced production of proinflammatory cytokines, including interleukin (IL)-12, IL-6, and tumor necrosis factor-alpha, as well as up-regulation of costimulatory molecules by CD8alpha(-) DCs, but not by CD8alpha(+) DCs, in vivo. Decreased DC activation profoundly influenced T helper (Th) cell responses, leading to impaired Th1 and enhanced Th2 responses. As a consequence of this immune modulation, host resistance to Leishmania major was compromised. We found that oxidized low-density lipoprotein (oxLDL) was the key active component responsible for this effect, as it could directly uncouple TLR-mediated signaling on CD8alpha(-) myeloid DCs and inhibit NF-kappaB nuclear translocation. These results show that a dyslipidemic microenvironment can directly interfere with DC responses to pathogen-derived signals and skew the development of T cell-mediated immunity.     

Induction of antigen-specific CD8+ cytotoxic T cells by dendritic cells co-electroporated with a dsRNA analogue and tumor antigen mRNA

The maturation state of dendritic cells (DCs) is an important determinant for the initiation and regulation of adaptive immune responses. In this study, we wanted to assess whether functional activation of human monocyte-derived DCs can be achieved by electroporation of an activation signal in the form of double-stranded (ds) RNA and whether simultaneous electroporation of the dsRNA with tumor antigen encoding mRNA can lead to the induction of a cytotoxic T-lymphocyte (CTL) response. Electroporation of immature DCs with poly(I:C(12)U), a dsRNA analogue, resulted in phenotypic as well as functional changes, indicative of DC maturation. Co-electroporation of DCs with both poly(I:C(12)U) and Melan-A/MART-1 encoding mRNA induced strong anti-Melan-A/MART-1 CD8(+) T-cell responses in vitro. Higher numbers of Melan-A/MART-1-specific CTLs were consistently obtained with poly(I:C(12)U)-activated DCs compared to DCs matured in the presence of an inflammatory cytokine cocktail. These results indicate that DC co-electroporation with both dsRNA and tumor antigen encoding mRNA induces fully activated and antigen-loaded DCs that promote antigen-specific CTL responses and may provide the basis for future immunotherapeutic strategies.     

CD8 positive T cells influence antigen-specific immune responses through the expression of chemokines.

The potential roles of CD8(+) T-cell-induced chemokines in the expansion of immune responses were examined using DNA immunogen constructs as model antigens. We coimmunized cDNA expression cassettes encoding the alpha-chemokines IL-8 and SDF-1alpha and the beta-chemokines MIP-1alpha, RANTES, and MCP-1 along with DNA immunogens and analyzed the resulting antigen-specific immune responses. In a manner more similar to the traditional immune modulatory role of CD4(+) T cells via the expression of Th1 or Th2 cytokines, CD8(+) T cells appeared to play an important role in immune expansion and effector function by producing chemokines. For instance, IL-8 was a strong inducer of CD4(+) T cells, indicated by strong T helper proliferative responses as well as an enhancement of antibody responses. MIP-1alpha had a dramatic effect on antibody responses and modulated the shift of immune responses to a Th2-type response. RANTES coimmunization enhanced the levels of antigen-specific Th1 and cytotoxic T lymphocyte (CTL) responses. Among the chemokines examined, MCP-1 was the most potent activator of CD8(+) CTL activity. The enhanced CTL results are supported by the increased expression of Th1 cytokines IFN-gamma and TNF-alpha and the reduction of IgG1/IgG2a ratio. Our results support that CD8(+) T cells may expand both humoral and cellular responses in vivo through the elaboration of specific chemokines at the peripheral site of infection during the effector stage of the immune response.     

CD4+ CD25+ regulatory T cells control T helper cell type 1 responses to foreign antigens induced by mature dendritic cells in vivo

Recent evidence suggests that in addition to their well known stimulatory properties, dendritic cells (DCs) may play a major role in peripheral tolerance. It is still unclear whether a distinct subtype or activation status of DC exists that promotes the differentiation of suppressor rather than effector T cells from naive precursors. In this work, we tested whether the naturally occurring CD4+ CD25+ regulatory T cells (Treg) may control immune responses induced by DCs in vivo. We characterized the immune response induced by adoptive transfer of antigen-pulsed mature DCs into mice depleted or not of CD25+ cells. We found that the development of major histocompatibility complex class I and II-restricted interferon gamma-producing cells was consistently enhanced in the absence of Treg. By contrast, T helper cell (Th)2 priming was down-regulated in the same conditions. This regulation was independent of interleukin 10 production by DCs. Of note, splenic DCs incubated in vitro with Toll-like receptor ligands (lipopolysaccharide or CpG) activated immune responses that remained sensitive to Treg function. Our data further show that mature DCs induced higher cytotoxic activity in CD25-depleted recipients as compared with untreated hosts. We conclude that Treg naturally exert a negative feedback mechanism on Th1-type responses induced by mature DCs in vivo.     

Organ-dependent in vivo priming of naive CD4+, but not CD8+, T cells by plasmacytoid dendritic cells

Plasmacytoid dendritic cells (PDCs) play a pivotal role as cytokine-secreting accessory cells in the antimicrobial immune defense. In contrast, the capacity of PDCs to act as antigen-presenting cells in naive T cell priming remains unclear. By studying T cell responses in mice that lack conventional DCs (cDCs), and by the use of a PDC-specific antigen-targeting strategy, we show that PDCs can initiate productive naive CD4(+) T cell responses in lymph nodes, but not in the spleen. PDC-triggered CD4(+) T cell responses differed from cDC-driven responses in that they were not associated with concomitant CD8(+) T cell priming. Our results establish PDCs as a bona fide DC subset that initiates unique CD4(+) Th cell-dominated primary immune responses.     

CD40L+ CD4+ memory T cells migrate in a CD62P-dependent fashion into reactive lymph nodes and license dendritic cells for T cell priming

There is growing evidence that the maturation state of dendritic cells (DCs) is a critical parameter determining the balance between tolerance and immunity. We report that mouse CD4⁺ effector memory T (T_EM) cells, but not naive or central memory T cells, constitutively expressed CD40L at levels sufficient to induce DC maturation in vitro and in vivo in the absence of antigenic stimulation. CD4⁺ T_EM cells were excluded from resting lymph nodes but migrated in a CD62P-dependent fashion into reactive lymph nodes that were induced to express CD62P, in a transient or sustained fashion, on high endothelial venules. Trafficking of CD4⁺ T_EM cells into chronic reactive lymph nodes maintained resident DCs in a mature state and promoted naive T cell responses and experimental autoimmune encephalomyelitis (EAE) to antigens administered in the absence of adjuvants. Antibodies to CD62P, which blocked CD4⁺ T_EM cell migration into reactive lymph nodes, inhibited DC maturation, T cell priming, and induction of EAE. These results show that T_EM cells can behave as endogenous adjuvants and suggest a mechanistic link between lymphocyte traffic in lymph nodes and induction of autoimmunity.     

Chemokines control naive CD8+ T cell selection of optimal lymph node antigen presenting cells

Naive antiviral CD8(+) T cells are activated in the draining LN (DLN) by dendritic cells (DCs) presenting viral antigens. However, many viruses infect LN macrophages, which participate in initiation of innate immunity and B cell activation. To better understand how and why T cells select infected DCs rather than macrophages, we performed intravital microscopy and ex vivo analyses after infecting mice with vaccinia virus (VV), a large DNA virus that infects both LN macrophages and DCs. Although CD8(+) T cells interact with both infected macrophages and DCs in the LN peripheral interfollicular region (PIR), DCs generate more frequent and stable interactions with T cells. VV infection induces rapid release of CCR5-binding chemokines in the LN, and administration of chemokine-neutralizing antibodies diminishes T cell activation by increasing T cell localization to macrophages in the macrophage-rich region (MRR) at the expense of PIR DCs. Similarly, DC ablation increases both T cell localization to the MRR and the duration of T cell-macrophage contacts, resulting in suboptimal T cell activation. Thus, virus-induced chemokines in DLNs enable antiviral CD8(+) T cells to distinguish DCs from macrophages to optimize T cell priming.