Splenic dendritic cell subsets prime and boost CD8 T cells and are involved in the generation of effector CD8 T cells

The ability of the dendritic cell (DC) subsets, CD8alpha+ and CD8alpha- DCs, to initiate a CD8 T cell response or to activate memory CD8 T cells and generate effector CD8 T cells has been controversial. In this study, we analyse the capacity of splenic DC subsets to induce CD8 T cell responses to a CD8 T cell epitope (pb9) of a malaria antigen. The administration of peptide-pulsed CD8alpha- or CD8alpha+ DCs primes and boosts a primed CD8 T cell response against the malaria epitope. In vitro, depletion of CD11c(+) DCs from mouse splenocytes, immunised with recombinant vaccinia virus Ankara (MVA) expressing pb9 epitope, significantly reduced the generation of pb9-specific IFNgamma producing effector CD8 T cells, indicating that splenic DCs are involved in the development of pb9-specific IFNgamma producing effector cells. Taken together, this result shows that both DC subsets have the ability to prime and boost CD8 T cell responses and are involved in the activation of memory CD8 T cells.     

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.     

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.     

CD40 ligand and CTLA-4 are reciprocally regulated in the Th1 cell proliferative response sustained by CD8(+) dendritic cells

Subsets of murine dendritic cells (DCs) from the spleen differ in their ability to induce proliferative responses in both primary and secondary CD4(+) T cells. Recent evidence indicates that lymphoid-related CD8(+) DCs fail to provide appropriate signals to freshly isolated secondary CD4(+) T cells to sustain their proliferation in vitro. In the present study, we examined peptide-pulsed CD8(-) and CD8(+) DCs for ability to stimulate Th1 and Th2 cell clones with the same Ag specificity. Defective ability to induce proliferation was selectively shown by CD8(+) DCs presenting Ag to the Th1 clone. The deficiency in CD8(+) DCs was overcome by CD40 triggering before peptide pulsing. When exposed to CD8(+) DCs in the absence of CD40 activation, the Th1 clone expressed low levels of CD40 ligand and high levels of surface CTLA-4. Neutralization of CTLA-4 during the DC/T cell coculture resulted in increased CD40 ligand expression and proliferation of T cells. Remarkably, the activation of CD40 on DCs under conditions that would increase Th1 cell proliferation, also resulted in down-regulation of surface CTLA-4. These results confirm differential effects of CD8(+) and CD8(-) DCs in the stimulation of Ag-primed Th cells. In addition, they suggest that reciprocal regulation of CD40 ligand and CTLA-4 expression occurs in Th1 cells exposed to CD8(+) DCs.     

IL-2 is required for the activation of memory CD8+ T cells via antigen cross-presentation

Dendritic cells (DCs) are capable of capturing exogenous Ag for the generation of MHC class I/peptide complexes. For efficient activation of memory CD8(+) T cells to occur via a cross-presentation pathway, DCs must receive helper signals from CD4(+) T cells. Using an in vitro system that reflects physiologic recall memory responses, we have evaluated signals that influence helper-dependent cross-priming, while focusing on the source and cellular target of such effector molecules. Concerning the interaction between CD4(+) T cells and DCs, we tested the hypothesis that CD40 engagement on DCs is critical for IL-12p70 (IL-12) production and subsequent stimulation of IFN-gamma release by CD8(+) T cells. Although CD40 engagement on DCs, or addition of exogenous IL-12 are both sufficient to overcome the lack of help, neither is essential. We next evaluated cytokines and chemokines produced during CD4(+) T cell/DC cross talk and observed high levels of IL-2 produced within the first 18-24 h of Ag-specific T cell engagement. Functional studies using blocking Abs to CD25 completely abrogated IFN-gamma production by the CD8(+) T cells. Although required, addition of exogenous IL-2 did not itself confer signals sufficient to overcome the lack of CD4(+) T cell help. Thus, these data support a combined role for Ag-specific, cognate interactions at the CD4(+) T cell/DC as well as the DC/CD8(+) T cell interface, with the helper effect mediated by soluble noncognate signals.     

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.     

Induction of CTL and nonpolarized Th cell responses by CD8alpha(+) and CD8alpha(-) dendritic cells

Two distinct dendritic cell (DC) subpopulations have been evidenced in mice on the basis of their differential CD8alpha expression and their localization in lymphoid organs. Several reports suggest that CD8alpha(+) and CD8alpha(-) DC subsets could be functionally different. In this study, using a panel of MHC class I- and/or class II-restricted peptides, we analyzed CD4(+) and CD8(+) T cell responses obtained after i.v. injection of freshly purified peptide-pulsed DC subsets. First, we showed that both DC subsets efficiently induce specific CTL responses and Th1 cytokine production in the absence of CD4(+) T cell priming. Second, we showed that in vivo activation of CD4(+) T cells by CD8alpha(+) or CD8alpha(-) DC, injected i.v., leads to a nonpolarized Th response with production of both Th1 and Th2 cytokines. The CD8alpha(-) subset induced a higher production of Th2 cytokines such as IL-4 and IL-10 than the CD8alpha(+) subset. However, IL-5 was produced by CD4(+) T cells activated by both DC subsets. When both CD4(+) and CD8(+) T cells were primed by DC injected i.v., a similar pattern of cytokines was observed, but, under these conditions, Th1 cytokines were mainly produced by CD8(+) T cells, while Th2 cytokines were produced by CD4(+) T cells. Thus, this study clearly shows that CD4(+) T cell responses do not influence the development of specific CD8(+) T cell cytotoxic responses induced either by CD8alpha(+) or CD8alpha(-) DC subsets.     

Provision of 4-1BB ligand enhances effector and memory CTL responses generated by immunization with dendritic cells expressing a human tumor-associated antigen

Up-regulation of receptor-ligand pairs during interaction of an MHC-presented epitope on dendritic cells (DCs) with cognate TCR may amplify, sustain, and drive diversity in the ensuing T cell immune response. Members of the TNF ligand superfamily and the TNFR superfamily contribute to this costimulatory molecule signaling. In this study, we used replication deficient adenoviruses to introduce a model tumor-associated Ag (the E7 oncoprotein of human papillomavirus 16) and the T cell costimulatory molecule 4-1BBL into murine DCs, and monitored the ability of these recombinant DCs to elicit E7-directed T cell responses following immunization. Splenocytes from mice immunized with DCs expressing E7 alone elicited E7-directed effector and memory CTL responses. Coexpression of 4-1BBL in these E7-expressing DCs increased effector and memory CTL responses when they were used for immunization. 4-1BBL expression up-regulated CD80 and CD86 second signaling molecules in DCs. We also report an additive effect of 4-1BBL and receptor activator of NF-kappa B/receptor activator of NF-kappa B ligand coexpression in E7-transduced DC immunogens on E7-directed effector and memory CTL responses and on MHC class II and CD80/86 expression in DCs. Additionally, expression of 4-1BBL in E7-transduced DCs reduced nonspecific T cell activation characteristic of adenovirus vector-associated immunization. The results have generic implications for improved or tumor Ag-expressing DC vaccines by incorporation of exogenous 4-1BBL. There are also specific implications for an improved DC-based vaccine for human papillomavirus 16-associated cervical carcinoma.     

Human dendritic cells sequentially matured with CD4(+) T cells as a secondary signal favor CTL and long-term T memory cell responses

Dendritic cells (DCs) are professional antigen-presenting cells involved in the control and initiation of immune responses. In vivo, DCs exposed at the periphery to maturation stimuli migrate to lymph nodes, where they receive secondary signals from CD4+ T helper cells. These DCs become able to initiate CD8+ cytotoxic T lymphocyte (CTL) responses. However, in vitro investigations concerning human monocyte-derived DCs have never focused on their functional properties after such sequential maturation. Here, we studied human DC phenotypes and functions according to this sequential exposure to maturation stimuli. As first signals, we used TNF-α/polyI:C mimicking inflammatory and pathogen stimuli and, as second signals, we compared activated CD4+ T helper cells to a combination of CD40-L/ IFN-γ. Our results show that a sequential activation with activated CD4+ T cells dramatically increased the maturation of DCs in terms of their phenotype and cytokine secretion compared to DCs activated with maturation stimuli delivered simultaneously. Furthermore, this sequential maturation led to the induction of CTL with a long-term effector and central memory phenotypes. Thus, sequential delivery of maturation stimuli, which includes CD4+ T cells, should be considered in the future to improve the induction of long-term CTL memory in DC-based immunotherapy.     

Inhibition of myeloid dendritic cell accessory cell function and induction of T cell anergy by alcohol correlates with decreased IL-12 production

Alcohol consumption inhibits accessory cell function and Ag-specific T cell responses. Myeloid dendritic cells (DCs) coordinate innate immune responses and T cell activation. In this report, we found that in vivo moderate alcohol intake (0.8 g/kg of body weight) in normal volunteers inhibited DC allostimulatory capacity. Furthermore, in vitro alcohol treatment during DC differentiation significantly reduced allostimulatory activity in a MLR using naive CD4(+) T cells, and inhibited tetanus toxoid Ag presentation by DCs. Alcohol-treated DCs showed reduced IL-12, increased IL-10 production, and a decrease in expression of the costimulatory molecules CD80 and CD86. Addition of exogenous IL-12 and IL-2, but not neutralization of IL-10, during MLR ameliorated the reduced allostimulatory capacity of alcohol-treated DCs. Naive CD4(+) T cells primed with alcohol-treated DCs showed decreased IFN-gamma production that was restored by exogenous IL-12, indicating inhibition of Th1 responses. Furthermore, CD4(+) T cells primed with alcohol-treated DCs were hyporesponsive to subsequent stimulation with the same donor-derived normal DCs, suggesting the ability of alcohol-treated DCs to induce T cell anergy. LPS-induced maturation of alcohol-treated immature DCs partially restored the reduced allostimulatory activity, whereas alcohol given only during DC maturation failed to inhibit DC functions, suggesting that alcohol primarily impairs DC differentiation rather than maturation. NFkappaB activation, a marker of DC maturation was not affected by alcohol. Taken together, alcohol both in vitro and in vivo can impair generation of Th1 immune responses via inhibition of DC differentiation and accessory cell function through mechanisms that involve decreased IL-12 induction.     

Murine CD4+ T cell responses are inhibited by cytotoxic T cell-mediated killing of dendritic cells and are restored by antigen transfer

Cytotoxic T lymphocytes (CTL) provide protection against pathogens and tumors. In addition, experiments in mouse models have shown that CTL can also kill antigen-presenting dendritic cells (DC), reducing their ability to activate primary and secondary CD8(+) T cell responses. In contrast, the effects of CTL-mediated killing on CD4(+) T cell responses have not been fully investigated. Here we use adoptive transfer of TCR transgenic T cells and DC immunization to show that specific CTL significantly inhibited CD4(+) T cell proliferation induced by DC loaded with peptide or low concentrations of protein antigen. In contrast, CTL had little effect on CD4(+) T cell proliferation induced by DC loaded with high protein concentrations or expressing antigen endogenously, even if these DC were efficiently killed and failed to accumulate in the lymph node (LN). Residual CD4(+) T cell proliferation was due to the transfer of antigen from carrier DC to host APC, and predominantly involved skin DC populations. Importantly, the proliferating CD4(+) T cells also developed into IFN-γ producing memory cells, a property normally requiring direct presentation by activated DC. Thus, CTL-mediated DC killing can inhibit CD4(+) T cell proliferation, with the extent of inhibition being determined by the form and amount of antigen used to load DC. In the presence of high antigen concentrations, antigen transfer to host DC enables the generation of CD4(+) T cell responses regardless of DC killing, and suggests mechanisms whereby CD4(+) T cell responses can be amplified.     

Prolonged antitumor NK cell reactivity elicited by CXCL10-expressing dendritic cells licensed by CD40L+ CD4+ memory T cells

Immunotherapy using dendritic cells (DCs) has the potential to activate both T cells and NK cells. We previously demonstrated the long-lasting antitumor responses by NK cells following immunization with bone marrow-derived DCs. In the current study, we demonstrate that long-term antitumor NK responses require endogenous DCs and a subset of effector memory CD4(+) T (CD4(+) T(EM)) cells. One month after DC immunization, injection of a tumor into DC-immunized mice leads to an increase in the expression of CXCL10 by endogenous DCs, thus directing NK cells into the white pulp where the endogenous DCs bridged CD4(+) T(EM) cells and NK cells. In this interaction, CD4(+) T(EM) cells express CD40L, which matures the endogenous DCs, and produce cytokines, such as IL-2, which activates NK cells. These findings suggest that DC vaccination can sustain long-term innate NK cell immunity but requires the participation of the adaptive immune system.     

Increased B7-H1 expression on dendritic cells correlates with programmed death 1 expression on T cells in simian immunodeficiency virus-infected macaques and may contribute to T cell dysfunction and disease progression

Suppression of dendritic cell (DC) function in HIV-1 infection is thought to contribute to inhibition of immune responses and disease progression, but the mechanism of this suppression remains undetermined. Using the rhesus macaque model, we show B7-H1 (programmed death [PD]-L1) is expressed on lymphoid and mucosal DCs (both myeloid DCs and plasmacytoid DCs), and its expression significantly increases after SIV infection. Meanwhile, its receptor, PD-1, is upregulated on T cells in both peripheral and mucosal tissues and maintained at high levels on SIV-specific CD8(+) T cell clones in chronic infection. However, both B7-H1 and PD-1 expression in SIV controllers was similar to that of controls. Expression of B7-H1 on both peripheral myeloid DCs and plasmacytoid DCs positively correlated with levels of PD-1 on circulating CD4(+) and CD8(+) T cells, viremia, and declining peripheral CD4(+) T cell levels in SIV-infected macaques. Importantly, blocking DC B7-H1 interaction with PD-1(+) T cells could restore SIV-specific CD4(+) and CD8(+) T cell function as evidenced by increased cytokine secretion and proliferative capacity. Combined, the results indicate that interaction of B7-H1-PD-1 between APCs and T cells correlates with impairment of CD4(+) Th cells and CTL responses in vivo, and all are associated with disease progression in SIV infection. Blockade of this pathway may have therapeutic implications for HIV-infected patients.     

A marked reduction in priming of cytotoxic CD8+ T cells mediated by stress-induced glucocorticoids involves multiple deficiencies in cross-presentation by dendritic cells

Protracted psychological stress elevates circulating glucocorticoids, which can suppress CD8(+) T cell-mediated immunity, but the mechanisms are incompletely understood. Dendritic cells (DCs), required for initiating CTL responses, are vulnerable to stress/corticosterone, which can contribute to diminished CTL responses. Cross-priming of CD8(+) T cells by DCs is required for initiating CTL responses against many intracellular pathogens that do not infect DCs. We examined the effects of stress/corticosterone on MHC class I (MHC I) cross-presentation and priming and show that stress/corticosterone-exposed DCs have a reduced ability to cross-present OVA and activate MHC I-OVA(257-264)-specific T cells. Using a murine model of psychological stress and OVA-loaded β(2)-microglobulin knockout "donor" cells that cannot present Ag, DCs from stressed mice induced markedly less Ag-specific CTL proliferation in a glucocorticoid receptor-dependent manner, and endogenous in vivo T cell cytolytic activity generated by cross-presented Ag was greatly diminished. These deficits in cross-presentation/priming were not due to altered Ag donation, Ag uptake (phagocytosis, receptor-mediated endocytosis, or fluid-phase uptake), or costimulatory molecule expression by DCs. However, proteasome activity in corticosterone-treated DCs or splenic DCs from stressed mice was partially suppressed, which limits formation of antigenic peptide-MHC I complexes. In addition, the lymphoid tissue-resident CD11b(-)CD24(+)CD8α(+) DC subset, which carries out cross-presentation/priming, was preferentially depleted in stressed mice. At the same time, CD11b(-)CD24(+)CD8α(-) DC precursors were increased, suggesting a block in development of CD8α(+) DCs. Therefore, glucocorticoid-induced changes in both the cellular composition of the immune system and intracellular protein degradation contribute to impaired CTL priming in stressed mice.     

Antigen presentation by an immature myeloid dendritic cell line does not cause CTL deletion in vivo, but generates CD8+ central memory-like T cells that can be rescued for full effector function

Immature dendritic cells (DC), in contrast to their mature counterparts, are incapable of mobilizing a CD8+ CTL response, and, instead, have been reported to induce CTL tolerance. We directly addressed the impact of immature vs mature DC on CTL responses by infusing adenovirus peptide-loaded DC (of the D1 cell line) into mice that had received adenovirus-specific naive TCR-transgenic CD8+ T cells. Whereas i.v. injection of mature DC triggered vigorous CTL expansion, immature DC elicited little proliferation involving only a minority of the TCR-transgenic CTL. Even though the latter CTL developed effector functions, including cytolytic activity and proinflammatory cytokine secretion, these cells differed significantly from CTL primed by mature DC in that they did not exhibit down-regulation of CD62L and CCR7, receptors involved in trapping of T cells in the lymphoid organs. Interestingly, adoptive transfer of CTL effector cells harvested after priming by either mature or immature DC into naive recipient mice, followed by exposure to adenovirus, yielded quantitatively and qualitatively indistinguishable CTL memory responses. Therefore, in vivo priming of naive CD8+ T cells by immature DC, although failing to induce a full-blown, systemic CTL response, resulted in the formation of central memory-like T cells that were able to expand and produce IFN-gamma upon secondary antigenic stimulation.     

Dendritic cells and CD28 costimulation are required to sustain virus-specific CD8+ T cell responses during the effector phase in vivo

Although much is known about the initiation of immune responses, much less is known about what controls the effector phase. CD8(+) T cell responses are believed to be programmed in lymph nodes during priming without any further contribution by dendritic cells (DCs) and Ag. In this study, we report the requirement for DCs, Ag, and CD28 costimulation during the effector phase of the CD8(+) T cell response. Depleting DCs or blocking CD28 after day 6 of primary influenza A virus infection decreases the virus-specific CD8(+) T cell response by inducing apoptosis, and this results in decreased viral clearance. Furthermore, effector CD8(+) T cells adoptively transferred during the effector phase fail to expand without DC, CD28 costimulation, and cognate Ag. The absence of costimulation also leads to reduced survival of virus-specific effector cells as they undergo apoptosis mediated by the proapoptotic molecule Bim. Finally, IL-2 treatment restored the effector response in the absence of CD28 costimulation. Thus, in contrast to naive CD8(+) T cells, which undergo an initial Ag-independent proliferation, effector CD8(+) T cells expanding in the lungs during the effector phase require Ag, CD28 costimulation, and DCs for survival and expansion. These requirements would greatly impair effector responses against viruses and tumors that are known to inhibit DC maturation and in chronic infections and aging where CD28(-/-) CD8(+) T cells accumulate.     

ISCOMATRIX adjuvant combines immune activation with antigen delivery to dendritic cells in vivo leading to effective cross-priming of CD8+ T cells

Cancer vaccines aim to induce CTL responses against tumors. Challenges for vaccine design are targeting Ag to dendritic cells (DCs) in vivo, facilitating cross-presentation, and conditioning the microenvironment for Th1 type immune responses. In this study, we report that ISCOM vaccines, which consist of ISCOMATRIX adjuvant and protein Ag, meet these challenges. Subcutaneous injection of an ISCOM vaccine in mice led to a substantial influx and activation of innate and adaptive immune effector cells in vaccine site-draining lymph nodes (VDLNs) as well as IFN-γ production by NK and NKT cells. Moreover, an ISCOM vaccine containing the model Ag OVA (OVA/ISCOM vaccine) was efficiently taken up by CD8α(+) DCs in VDLNs and induced their maturation and IL-12 production. Adoptive transfer of transgenic OT-I T cells revealed highly efficient cross-presentation of the OVA/ISCOM vaccine in vivo, whereas cross-presentation of soluble OVA was poor even at a 100-fold higher concentration. Cross-presenting activity was restricted to CD8α(+) DCs in VDLNs, whereas Langerin(+) DCs and CD8α(-) DCs were dispensable. Remarkably, compared with other adjuvant systems, the OVA/ISCOM vaccine induced a high frequency of OVA-specific CTLs capable of tumor cell killing in different tumor models. Thus, ISCOM vaccines combine potent immune activation with Ag delivery to CD8α(+) DCs in vivo for efficient induction of CTL responses.     

Flt3 ligand-generated murine plasmacytoid and conventional dendritic cells differ in their capacity to prime naive CD8 T cells and to generate memory cells in vivo

Mature dendritic cells (DCs) have the capacity to induce efficient primary T cell response and effector cell differentiation. Thus, these cells are a major tool in the design of various immunotherapeutic protocols. We have tested the capacity of different subsets of matured DCs pulsed with a peptide to induce the differentiation of naive CD8 T cells into memory cells in vivo. Flt3 ligand (FL) induces the differentiation of conventional DCs (cDCs) and plasmacytoid DCs (PDCs) from murine bone marrow precursors in vitro. After maturation, both subsets become strong stimulators of Ag-specific T cell responses in vitro. However, the in vivo T cell stimulatory capacity of these DC subsets has not been studied in detail. In the present study, we demonstrate that mature FL-generated DCs induce efficient peptide-specific CD8 T cell response and memory cell differentiation in vivo. This is mainly due to the cDC subset because the PDC subset induced only a negligible primary CD8 response without detectable levels of memory CD8 T cell differentiation. Thus, in vitro FL-generated mature cDCs, but not PDCs, are potent stimulators of peptide-specific CD8 T cell responses and memory generation in vivo.     

Induction of hyper Th1 cell-type immune responses by dendritic cells lacking the suppressor of cytokine signaling-1 gene

Suppressor of cytokine signaling (SOCS1/JAB) has been shown to play an important role in regulating dendritic cell (DC) function and suppressing inflammatory diseases and systemic autoimmunity. However, role of SOCS1 in DCs for the initiation of Th cell response has not been clarified. Here we demonstrate that SOCS1-deficient DCs induce stronger Th1-type responses both in vitro and in vivo. SOCS1-deficient DCs induced higher IFN-gamma production from naive T cells than wild-type (WT) DCs in vitro. Lymph node T cells also produced a higher amount of IFN-gamma when SOCS1-deficient bone marrow-derived DCs (BMDCs) were transferred in vivo. Moreover, SOCS1(-/-) BMDCs raised more effective anti-tumor immunity than WT BMDCs. Microarray analysis revealed that IFN-inducible genes were highly expressed in SOCS1-deficient DCs without IFN stimulation, suggesting hyper STAT1 activation in SOCS1(-/-) DCs. These phenotypes of SOCS1-deficient DCs were similar to those of CD8alpha(+) DCs, and in the WT spleen, SOCS1 is expressed at higher levels in the Th2-inducing CD4(+) DC subset, relative to the Th1-inducing CD8alpha(+) DC subset. We propose that reduction of the SOCS1 gene expression in DCs leads to CD8alpha(+) DC-like phenotype which promotes Th1-type hyperresponses.     

The generation of both T killer and Th cell clones specific for the tumor-associated antigen HER2 using retrovirally transduced dendritic cells

Induction of antitumor immunity involves the presence of both CD8(+) CTLs and CD4(+) Th cells specific for tumor-associated Ags. Attempts to eradicate cancer by adoptive T cell transfer have been limited due to the difficulty of generating T cells with defined Ag specificity. The current study focuses on the generation of CTL and Th cells against the tumor-associated Ag HER2 using autologous dendritic cells (DC) derived from CD34(+) hematopoietic progenitor cells which have been retrovirally transduced with the human epidermal growth factor receptor 2 (HER2) gene. HER2-transduced DC elicited HER2-specific CD8(+) CTL that lyse HER2-overexpressing tumor cells in context of distinct HLA class I alleles. The induction of both HLA-A2 and -A3-restricted HER2-specific CTL was verified on a clonal level. In addition, retrovirally transduced DC induced CD4(+) Th1 cells recognizing HER2 in context with HLA class II. HLA-DR-restricted CD4(+) T cells were cloned that released IFN-gamma upon stimulation with DC pulsed with the recombinant protein of the extracellular domain of HER2. These data indicate that retrovirally transduced DC expressing the HER2 molecule present multiple peptide epitopes and subsequently elicit HER2-specific CTL and Th1 cells. The method of stimulating HER2-specific CD8(+) and CD4(+) T cells with retrovirally transduced DC was successfully implemented for generating HER2-specific CTL and Th1 clones from a patient with HER2-overexpressing breast cancer. The ability to generate and expand HER2-specific, HLA-restricted CTL and Th1 clones in vitro facilitates the development of immunotherapy regimens, in particular the adoptive transfer of both autologous HER2-specific T cell clones in patients with HER2-overexpressing tumors without the requirement of defining immunogenic peptides.