Dynamic control of self-specific CD8+ T cell responses via a combination of signals mediated by dendritic cells

It is acknowledged that T cell interactions with mature dendritic cells (DC) lead to immunity, whereas interactions with immature DC lead to tolerance induction. Using a transgenic murine system, we have examined how DC expressing self-peptides control naive, self-reactive CD8+ T cell responses in vitro and in vivo. We have shown, for the first time, that immature DC can also stimulate productive activation of naive self-specific CD8+ T cells, which results in extensive proliferation, the expression of a highly activated cell surface phenotype, and differentiation into autoimmune CTL. Conversely, mature DC can induce abortive activation of naive CD8+ T cells, which is characterized by low-level proliferation, the expression of a partially activated cell surface phenotype which does not result in autoimmune CTL. Critically, both CD8+ T cell responses are determined by a combination of signals mediated by the DC, and that altering any one of these signals dramatically shifts the balance between autoimmunity and self-tolerance induction. We hypothesize that DC maintain the steady state of self-tolerance among self-specific CD8+ T cells in an active and dynamic manner, licensing productive immune responses against self-tissues only when required.     

Requirement of mature dendritic cells for efficient activation of influenza A-specific memory CD8+ T cells

It is critical to identify the developmental stage of dendritic cells (DCs) that is most efficient at inducing CD8+ T cell responses. Immature DCs can be generated from monocytes with GM-CSF and IL-4, while maturation is accomplished by the addition of stimuli such as monocyte-conditioned medium, CD40 ligand, and LPS. We evaluated the ability of human monocytes and immature and mature DCs to induce CD8+ effector responses to influenza virus Ags from resting memory cells. We studied replicating virus, nonreplicating virus, and the HLA-A*0201-restricted influenza matrix protein peptide. Sensitive and quantitative assays were used to measure influenza A-specific immune responses, including MHC class I tetramer binding assays, enzyme-linked immunospot assays for IFN-gamma production, and generation of cytotoxic T cells. Mature DCs were demonstrated to be superior to immature DC in eliciting IFN-gamma production from CD8+ effector cells. Furthermore, only mature DCs, not immature DCs, could expand and differentiate CTL precursors into cytotoxic effector cells over 7 days. An exception to this was immature DCs infected with live influenza virus, because of the virus's known maturation effect. Finally, mature DCs pulsed with matrix peptide induced CTLs from highly purified CD8+ T cells without requiring CD4+ T cell help. These differences between DC stages were independent of Ag concentrations or the number of immature DCs. In contrast to DCs, monocytes were markedly inferior or completely ineffective stimulators of T cell immunity. Our data with several qualitatively different assays of the memory CD8+ T cell response suggest that mature cells should be considered as immunotherapeutic adjuvants for Ag delivery.     

Exosomes as potent cell-free peptide-based vaccine. I. Dendritic cell-derived exosomes transfer functional MHC class I/peptide complexes to dendritic cells

Current immunization protocols in cancer patients involve CTL-defined tumor peptides. Mature dendritic cells (DC) are the most potent APCs for the priming of naive CD8(+) T cells, eventually leading to tumor eradication. Because DC can secrete MHC class I-bearing exosomes, we addressed whether exosomes pulsed with synthetic peptides could subserve the DC function consisting in MHC class I-restricted, peptide-specific CTL priming in vitro and in vivo. The priming of CTL restricted by HLA-A2 molecules and specific for melanoma peptides was performed: 1) using in vitro stimulations of total blood lymphocytes with autologous DC pulsed with GMP-manufactured autologous exosomes in a series of normal volunteers; 2) in HLA-A2 transgenic mice (HHD2) using exosomes harboring functional HLA-A2/Mart1 peptide complexes. In this study, we show that: 1). DC release abundant MHC class I/peptide complexes transferred within exosomes to other naive DC for efficient CD8(+) T cell priming in vitro; 2). exosomes require nature's adjuvants (mature DC) to efficiently promote the differentiation of melanoma-specific effector T lymphocytes producing IFN-gamma (Tc1) effector lymphocytes in HLA-A2 transgenic mice (HHD2). These data imply that exosomes might be a transfer mechanism of functional MHC class I/peptide complexes to DC for efficient CTL activation in vivo.     

NKT cells provide help for dendritic cell-dependent priming of MHC class I-restricted CD8+ T cells in vivo

Dendritic cells (DC) are potent APCs for naive T cells in vivo. This is evident by inducing T cell responses through adoptive DC transfer. Priming specific CTL responses in vivo often requires "help". We study alternative sources of help in DC-dependent priming of MHC class I-restricted CTL. Priming an anti-viral CTL response in naive B6 mice by adoptive transfer of antigenic peptide-pulsed DC required CD4(+) T cell help. CTL priming was facilitated by providing MHC class II-dependent specific help. Furthermore, transfers of MHC class II-deficient pulsed DC into naive, normal hosts, or DC transfers into naive, CD4(+) T cell-depleted hosts primed CTL inefficiently. Pretreatment of DC with immune-stimulating oligodeoxynucleotides rendered them more efficient for CD4(+) T cell-independent priming of CTL. DC copresenting a K(b)-binding antigenic peptide and the CD1d-binding glycolipid alpha-galactosyl-ceramide efficiently primed CTL in a class II-independent way. To obtain NKT cell-dependent help in CTL priming, the same DC had to present both the peptide and the glycolipid. CTL priming by adoptive DC transfer was largely NK cell-dependent. The requirement for NK cells was only partially overcome by recruiting NKT cell help into DC-dependent CTL priming. NKT cells thus are potent helper cells for DC-dependent CTL priming.     

Steady state dendritic cells present parenchymal self-antigen and contribute to, but are not essential for, tolerization of naive and Th1 effector CD4 cells

Bone marrow-derived APC are critical for both priming effector/memory T cell responses to pathogens and inducing peripheral tolerance in self-reactive T cells. In particular, dendritic cells (DC) can acquire peripheral self-Ags under steady state conditions and are thought to present them to cognate T cells in a default tolerogenic manner, whereas exposure to pathogen-associated inflammatory mediators during the acquisition of pathogen-derived Ags appears to reprogram DCs to prime effector and memory T cell function. Recent studies have confirmed the critical role of DCs in priming CD8 cell effector responses to certain pathogens, although the necessity of steady state DCs in programming T cell tolerance to peripheral self-Ags has not been directly tested. In the current study, the role of steady state DCs in programming self-reactive CD4 cell peripheral tolerance was assessed by combining the CD11c-diphtheria toxin receptor transgenic system, in which DC can be depleted via treatment with diphtheria toxin, with a TCR-transgenic adoptive transfer system in which either naive or Th1 effector CD4 cells are induced to undergo tolerization after exposure to cognate parenchymally derived self-Ag. Although steady state DCs present parenchymal self-Ag and contribute to the tolerization of cognate naive and Th1 effector CD4 cells, they are not essential, indicating the involvement of a non-DC tolerogenic APC population(s). Tolerogenic APCs, however, do not require the cooperation of CD4(+)CD25(+) regulatory T cells. Similarly, DC were required for maximal priming of naive CD4 cells to vaccinia viral-Ag, but priming could still occur in the absence of DC.     

Induction of primary human CD8+ T lymphocyte responses in vitro using dendritic cells

The ability of two different human professional APCs, specifically macrophages (Mphi) and dendritic cells (DC), to stimulate primary responses in human CD8+ T lymphocytes was examined using both allogeneic and Ag-pulsed autologous APCs. CTL responses in CD8+ T lymphocytes isolated from HIV-uninfected donors were evaluated against six different HIV epitopes that are restricted by four different HLA alleles using autologous human PBMC-derived Mphi and DCs for primary stimulation. In a side-by-side experiment, immature DCs, but not Mphi, were able to prime a CTL response against the B14-restricted p24gag 298-306 epitope; mature DCs were also able to prime a response against this epitope. In addition, DCs were capable of priming CD8+ CTL responses against the B8-restricted p24gag 259-267 epitope. In contrast, Mphi were unable to prime strong CTL responses against other epitopes. Since the Ag-specific cytotoxic responses required subsequent rounds of restimulation before they could be detected, the ability of the allogeneic Mphi and DCs to directly prime CD8+ T lymphocyte responses without subsequent restimulation was examined. Similar to the aforementioned peptide-specific results, DCs were more efficient than Mphi in priming both allogeneic proliferative and cytotoxic responses in human CD8+ T lymphocytes. Collectively, these results promote an enhanced status for DCs in the primary stimulation of human CD8+ T lymphocytes.     

Paternal antigen-bearing cells transferred during insemination do not stimulate anti-paternal CD8+ T cells: role of estradiol in locally inhibiting CD8+ T cell responses

Maternal immunological tolerance of the semiallogeneic fetus involves several overlapping mechanisms to balance maternal immunity and fetal development. Anti-paternal CD8+ T cells are suppressed during pregnancy in some but not all mouse models. Since semen has been shown to mediate immune modulation, we tested whether exposure to paternal Ag during insemination activated or tolerized anti-paternal CD8+ T cells. The uterine lumen of mated female mice contained male MHC I+ cells that stimulated effector, but not naive, CD8+ T cells ex vivo. Maternal MHC class I+ myeloid cells fluxed into the uterine lumen in response to mating and cross-presented male H-Y Ag to effector, but not naive, CD8+ T cells ex vivo. However, neither unprimed nor previously primed TCR-transgenic CD8+ T cells specific for either paternal MHC I or H-Y Ag proliferated in vivo after mating. These T cells subsequently responded normally to i.p. challenge, implicating ignorance rather than anergy as the main reason for the lack of response. CD8+ T cells responded to either peptide Ag or male cells delivered intravaginally in ovariectomized mice, but this response was inhibited by systemic estradiol (inducing an estrus-like state). Subcutaneous Ag induced responses in both cases. Allogeneic dendritic cells did not induce responses intravaginally even in ovariectomized mice in the absence of estradiol. These results suggest that inhibition of antiallogeneic responses is restricted both locally to the reproductive tract and temporally to the estrous phase of the menstrual cycle, potentially decreasing the risk of maternal immunization against paternal Ags during insemination.     

Critical role of costimulation in the activation of naive antigen-specific TCR transgenic CD8+ T cells in vitro.

The influence of costimulation on the activation of naive CD8+ T cells and thymocytes was studied in vitro using H-Y-specific TCR-transgenic mice and H-Y antigenic peptide. Using a variety of physiological APC types, the activation of naive CD8+ T cells depended strictly on costimulation, which could not be substituted by high epitope density. T cell activation is known to be regulated by the interactions between CD86/CD80 and CD28/CD152, although it remains unclear whether the B7 isoforms have distinct roles. Addition of soluble anti-CD86 Ab led to profound inhibition of T cell reactivity, further confirming the importance of costimulation in naive CD8+ T cell activation. Finally, TCR engagement in the absence of costimulation had no effect on the subsequent reactivity of peripheral naive transgenic CD8+ T cells, but induced nonresponsiveness in mature CD8+ transgenic thymocytes. Collectively, these results demonstrate the importance of costimulation for naive CD8+ T cell activation, suggest that CD80 and CD86 can mediate opposing effects, possibly due to differential interaction with CD152 and CD28, and indicate differences in the sensitivity of immature vs mature CD8+ T cells to the induction of nonresponsiveness following costimulation-deficient Ag presentation.     

A new dynamic model of CD8+ T effector cell responses via CD4+ T helper-antigen-presenting cells

A long-standing paradox in cellular immunology has been the conditional requirement for CD4(+) Th cells in priming of CD8(+) CTL responses. We propose a new dynamic model of CD4(+) Th cells in priming of Th-dependent CD8(+) CTL responses. We demonstrate that OT II CD4(+) T cells activated by OVA-pulsed dendritic cells (DC(OVA)) are Th1 phenotype. They acquire the immune synapse-composed MHC II/OVAII peptide complexes and costimulatory molecules (CD54 and CD80) as well as the bystander MHC class I/OVAI peptide complexes from the DC(OVA) by DC(OVA) stimulation and thus also the potential to act themselves as APCs. These CD4(+) Th-APCs stimulate naive OT I CD8(+) T cell proliferation through signal 1 (MHC I/OVAI/TCR) and signal 2 (e.g., CD54/LFA-1 and CD80/CD28) interactions and IL-2 help. In vivo, they stimulate CD8(+) T cell proliferation and differentiation into CTLs and induce effective OVA-specific antitumor immunity. Taken together, this study demonstrates that CD4(+) Th cells carrying acquired DC Ag-presenting machinery can, by themselves, efficiently stimulate CTL responses. These results have substantial implications for research in antitumor and other aspects of immunity.     

CD8+ T cell-dependent elimination of dendritic cells in vivo limits the induction of antitumor immunity

The fate of dendritic cells (DC) after they have initiated a T cell immune response is still undefined. We have monitored the migration of DC labeled with a fluorescent tracer and injected s.c. into naive mice or into mice with an ongoing immune response. DC not loaded with Ag were detected in the draining lymph node in excess of 7 days after injection with maximum numbers detectable approximately 40 h after transfer. In contrast, DC that had been loaded with an MHC class I-binding peptide disappeared from the lymph node with kinetics that parallel the known kinetics of activation of CD8+ T cells to effector function. In the presence of high numbers of specific CTL precursors, as in TCR transgenic mice, DC numbers were significantly decreased by 72 h after injection. The rate of DC disappearance was extremely rapid and efficient in recently immunized mice and was slower in "memory" mice in which memory CD8+ cells needed to reacquire effector function before mediating DC elimination. We also show that CTL-mediated clearance of Ag-loaded DC has a notable effect on immune responses in vivo. Ag-specific CD8+ T cells failed to divide in response to Ag presented on a DC if the DC were targets of a pre-existing CTL response. The induction of antitumor immunity by tumor Ag-loaded DC was also impaired. Therefore, CTL-mediated clearance of Ag-loaded DC may serve as a negative feedback mechanism to limit the activity of DC within the lymph node.     

Reversal of CD8+ T cell ignorance and induction of anti-tumor immunity by peptide-pulsed APC

In the present report, we have studied the potential of naive and activated effector CD8(+) T cells to function as anti-tumor T cells to a solid tumor using OVA-specific T cells from TCR-transgenic OT-I mice. Adoptive transfer of naive OT-I T cells into tumor-bearing syngeneic mice did not inhibit tumor cell growth. The adoptively transferred OT-I T cells did not proliferate in lymphoid tissue of tumor-bearing mice and were not anergized by the tumor. In contrast, adoptive transfer of preactivated OT-I CTL inhibited tumor growth in a dose-dependent manner, indicating that E.G7 was susceptible to immune effector cells. Importantly, naive OT-I T cells proliferated and elicited an anti-tumor response if they were adoptively transferred into normal or CD4-deficient mice that were then vaccinated with GM-CSF-induced bone marrow-derived OVA-pulsed APC. Collectively, these data indicate that even though naive tumor-specific T cells are present at a relatively high fraction they remain ignorant of the tumor and demonstrate that a CD8-mediated anti-tumor response can be induced by Ag-pulsed APC without CD4 T cell help.     

mRNA transfection of DC in the immature or mature state: comparable in vitro priming of Th and cytotoxic T lymphocytes against DC electroporated with tumor cell line-derived mRNA

BACKGROUND: The use of mRNA in vaccine studies has generally been through loading or transfection of immature DC followed by a maturation step. A recent study has suggested that this strategy may result in inferior priming of cytotoxic T lymphocytes (CTL). Furthermore the study did not address any possible effects on the priming of CD4(+) T-cell responses. METHODS: We compared mRNA transfection of mature DC with that of immature DC, using as a read-out their capacity to prime autologous T cells during one cycle of in vitro stimulation. In this model system we used mRNA from the tumor cell line Jurkat E6. DC transfected at either the immature stage (day 5) or mature stage (day 7) displayed a similar phenotype. RESULTS: Interestingly, no major differences in their ability to prime CD4 and CD8 T-cell responses were observed. As in vitro priming to some extent may reflect the capacity of these DC to prime T cells in vivo after vaccination, these studies support the use of mRNA-transfected mature DC in clinical protocols. DISCUSSION: Transfection of DC at the end of the maturation process represents a logistical improvement in the GMP production of mRNA-transfected DC for clinical protocols.     

CD40 activation in vivo overcomes peptide-induced peripheral cytotoxic T-lymphocyte tolerance and augments anti-tumor vaccine efficacy.

The outcome of antigen recognition by naive CD8+ cytotoxic T lymphocytes (CTLs) in the periphery is orchestrated by CD4+ T-helper cells, and can either lead to priming or tolerization. The presence of T-helper cells favors the induction of CTL immunity, whereas the absence of T-helper cells can result in CTL tolerance. The action of T helper cells in CTL priming is mediated by CD40-CD40 ligand interactions. We demonstrate here that triggering of CD40 in vivo can considerably enhance the efficacy of peptide-based anti-tumor vaccines. The combination of a tolerogenic peptide vaccine containing a minimal essential CTL epitope with an activating antibody against CD40 converts tolerization into strong CTL priming. Moreover, CD40 ligation can provide an already protective tumor-specific peptide vaccine with the capacity to induce therapeutic CTL immunity in tumor-bearing mice. These findings indicate that the CD40-CD40 ligand pair can act as a 'switch', determining whether naive peripheral CTLs are primed or tolerized, and support the clinical use of CD40-stimulating agents as components of anti-cancer vaccines.     

Impact of CCR7 on priming and distribution of antiviral effector and memory CTL

The chemokine receptor CCR7 is a key factor in the coordinate migration of T cells and dendritic cells (DC) into and their localization within secondary lymphoid organs. In this study we investigated the impact of CCR7 on CD8(+) T cell responses by infecting CCR7(-/-) mice with lymphocytic choriomeningitis virus (LCMV). We found that the absence of CCR7 affects the magnitude of an antiviral CTL response during the acute phase, with reduced numbers of virus-specific CTL in all lymphoid and nonlymphoid organs tested. On the single cell level, CCR7-deficient CTL gained full effector function, such that antiviral protection in CCR7-deficient mice was complete, but delayed. Similarly, adoptive transfer experiments using DC from CCR7-deficient or competent mice for the priming of CCR7-positive or CCR7-negative CD8(+) T cells, respectively, revealed that ectopic positioning of DC and CTL outside organized T cell zones results in reduced priming efficacy. In the memory phase, CCR7-deficient mice maintained a stable LCMV-specific CTL population, predominantly in nonlymphoid organs, and rapidly mounted protective CTL responses against a challenge infection with a vaccinia virus recombinant for the gp33 epitope of LCMV. Taken together, the CCR7-dependent organization of the T cell zone does not appear to be a prerequisite for antiviral effector CTL differentiation and the sustenance of antiviral memory responses in lymphoid or peripheral tissues.     

Priming of CTLs by lymphocytic choriomeningitis virus depends on dendritic cells

Appropriate activation of naive CD8(+) T cells depends on the coordinated interaction of these cells with professional APC that present antigenic peptides in the context of MHC class I molecules. It is accepted that dendritic cells (DC) are efficient in activating naive T cells and are unique in their capacity to prime CD8(+) T cell responses against exogenous cell-associated Ags. Nevertheless, it is unclear whether epitopes, derived from endogenously synthesized proteins and presented by MHC class I molecules on the surface of other APC including B cells and macrophages, can activate naive CD8(+) T cells in vivo. By infecting transgenic CD11c-DTR/GFP mice that allow conditional depletion of DC with lymphocytic choriomeningitis virus (LCMV), which infects all types of APC and elicits a vigorous CTL response, we unambiguously show that priming of LCMV-specific CD8(+) T cells is crucially dependent on DC, despite ample presence of LCMV-infected macrophages and B cells in secondary lymphoid organs.     

Memory CD8+ T cells protect dendritic cells from CTL killing

CD8(+) T cells have been shown to be capable of either suppressing or promoting immune responses. To reconcile these contrasting regulatory functions, we compared the ability of human effector and memory CD8(+) T cells to regulate survival and functions of dendritic cells (DC). We report that, in sharp contrast to the effector cells (CTLs) that kill DCs in a granzyme B- and perforin-dependent mechanism, memory CD8(+) T cells enhance the ability of DCs to produce IL-12 and to induce functional Th1 and CTL responses in naive CD4(+) and CD8(+) T cell populations. Moreover, memory CD8(+) T cells that release the DC-activating factor TNF-alpha before the release of cytotoxic granules induce DC expression of an endogenous granzyme B inhibitor PI-9 and protect DCs from CTL killing with similar efficacy as CD4(+) Th cells. The currently identified DC-protective function of memory CD8(+) T cells helps to explain the phenomenon of CD8(+) T cell memory, reduced dependence of recall responses on CD4(+) T cell help, and the importance of delayed administration of booster doses of vaccines for the optimal outcome of immunization.     

Simultaneous induction of CD4 T cell tolerance and CD8 T cell immunity by semimature dendritic cells

Previous studies suggested that depending on their maturation state, dendritic cells (DC) could either induce T cell tolerance (immature and semimature DC) or T cell activation (mature DC). Pretreatment of C57BL/6 mice with encephalitogenic myelin oligodendrocyte glycoprotein (MOG)(35-55) peptide-loaded semimature DC protected from MOG-induced autoimmune encephalomyelitis. This protection was mediated by IL-10-producing CD4 T cells specific for the self Ag. Here we show that semimature DC loaded with the MHC class II-restricted nonself peptide Ag (OVA) induce an identical regulatory T cell cytokine pattern. However, semimature DC loaded simultaneously with MHC class II- and MHC class I-restricted peptides, could efficiently initiate CD8 T cell responses leading to autoimmune diabetes in a TCR-transgenic adoptive transfer model. Double-peptide-loaded semimature DC also induced simultaneously in the same animal partially activated CD8 T cells with cytolytic function as well as protection from MOG-induced autoimmune encephalomyelitis. Our study suggests that the decision between tolerance and immunity not only depends on the DC, but also on the type and activation requirements of the responding T cell.     

A spontaneously arising pancreatic tumor does not promote the differentiation of naive CD8+ T lymphocytes into effector CTL

In this report, we address whether a growing tumor provides sufficient inflammatory signals to promote activation, clonal expansion, and acquisition of effector functions by naive tumor-specific CD8(+) T lymphocytes. CD8(+) T lymphocytes obtained from hemagglutinin (HA)-specific clone 4 TCR-transgenic mice were injected into recipient mice that spontaneously develop pancreatic tumors expressing HA as a tumor-associated Ag (RIP-Tag2-HA mice). When 3 x 10(6) clone 4 CD8(+) T cells were transferred into tumor-bearing mice, the cells became activated in the pancreatic lymph nodes where they proliferated and acquired effector functions such as cytolytic activity and IFN-gamma production. Surprisingly, reducing the number of adoptively transferred CD8(+) T cells led to a parallel reduction in the proportion of the activated cells that exhibited effector functions, suggesting that CTL differentiation was induced by the large numbers of activated CD8(+) T cells and not the tumor environment. Provision of tumor-specific CD4(+) helper cells provided the signals required to promote both the development of CTL effector functions and increased clonal expansion, resulting in tumor eradication. Considering that only small numbers of tumor-specific CD8(+) T cells would be present in a conventional T cell repertoire, these data suggest that tumor growth alone may not provide the inflammatory signals necessary to support the development of CD8(+) T cell effector functions.     

Phenotypic and functional characterization of mouse hepatic CD8 alpha+ lymphoid-related dendritic cells

Recently, attention has focussed on phenotypic and functional differences between classic myeloid dendritic cells (DC), and DC that reportedly develop from an early, committed lymphoid precursor. In mice, DC from these separate hemopoietic lineages differ by their surface expression of CD8 alpha. We undertook a comparative study of CD8 alpha+ (CD11blow; lymphoid-related) and CD8 alpha- (CD11bhigh; myeloid) DC isolated from mouse liver. CD8 alpha+ and CD8 alpha- DC each constituted 相似文献   

In vivo induction of cytotoxic T cell response by a free synthetic peptide requires CD4+ T cell help.

Most attempts to induce CTL responses by in vivo priming with free synthetic peptides have been unsuccessful so far. However, two separate studies have recently succeeded in inducing antiviral CTL responses by immunizing mice with unmodified free synthetic peptides derived from nucleoproteins from either lymphocytic choriomeningitis virus or Sendai virus. In the present study, we have analyzed the cellular mechanisms by which the lymphocytic choriomeningitis virus synthetic peptide induced CTL responses. We demonstrated that this peptide, which was previously shown to be recognized by CD8+ T cells, also contains a helper CD4+ T cell epitope. It stimulates in vivo both CD4+ T cell-mediated CTL response. The in vivo elimination of CD4+ T cells by treatment with a mAb was shown to strongly reduce the antipeptide CTL response. This study therefore demonstrates that to be able to induce CTL responses, a peptide has to stimulate both CD4+ and CD8+ T cell subset.