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Summary: Despite recent therapeutic advances, including theintroduction of novel cytostatic drugs and therapeutic antibodies,many cancer patients will experience recurrent or metastaticdisease. Current treatment options, particularly for those patientswith metastatic breast, prostate, or skin cancers, are complex andhave limited curative potential. Recent clinical trials, however, haveshown that cell-based therapeutic vaccines may be used to generatebroad-based, antitumor immune responses. Dendritic cells (DC)have proved to be the most efficacious cellular component fortherapeutic vaccines, serving as both the adjuvant and antigendelivery vehicle. At present it is not possible to noninvasivelydetermine the fate of DC-based vaccines after their administrationto human subjects. In this study, we demonstrate that in vitro-generated mouse DC can be readily labeled with superparamag-netic iron oxide nanoparticles, Feridex, without altering cellmorphology, or their phenotypic and functional maturation.Feridex-labeling enables the detection of DC in vivo after theirmigration to draining lymph nodes using a 1.5T clinical magneticresonance scanner. In addition, we report a semiquantitativeapproach for analysis of magnetic resonance images and show thatthe Feridex-induced signal void volume, and fractional signal loss,correlates with the delivery and migration of small numbers of invitro-generated DC. These findings, together with ongoingpreclinical studies, are key to gaining information critical forimproving the efficacy of therapeutic vaccines for the treatmentcancer, and potentially, chronic infectious diseases.Key Words: immunotherapy, dendritic cells, vaccine, magneticresonance imaging, cancer

Although typically considered a neurotransmitter, there is substantial evidence that serotonin (5-HT) plays an important role in the pathogenesis of inflammatory disorders. Despite these findings, the precise role of 5-HT in modulating immune function, particularly T-cell function, remains elusive. We report that naive T cells predominantly express the type 7 5-HT receptor (5-HTR), and expression of this protein is substantially enhanced on T-cell activation. In addition, T-cell activation leads to expression of the 5-HT1B and 5-HT2A receptors. Significantly, exogenous 5-HT induces rapid phosphorylation of extracellular signal-regulated kinase-1 and -2 (ERK1/2) and IκBα in naive T cells. 5-HT–induced activation of ERK1/2 and NFκB is inhibited by preincubation with a specific 5-HT7 receptor antagonist. Thus, 5-HT signaling via the 5-HT7 receptor may contribute to early T-cell activation. In turn, 5-HT synthesized by T cells may act as an autocrine factor. Consistent with this hypothesis, we found that inhibition of 5-HT synthesis with parachlorophenylalanine (PCPA) impairs T-cell activation and proliferation. Combined, these data demonstrate a fundamental role for 5-HT as an intrinsic cofactor in T-cell activation and function and suggest an alternative mechanism through which immune function may be regulated by indoleamine 2,3-dioxygenase–mediated catabolism of tryptophan.

We determined whether distinct subclasses of dendritic cells (DC) could polarize cytokine production and regulate the pattern of xenograft rejection. C57BL/6 recipients, transplanted with Lewis rat hearts, exhibited a predominantly CD11c+CD8α+ splenic DC population and an intragraft cytokine profile characteristic of a Th1-dominant response. In contrast, BALB/c recipients of Lewis rat heart xenografts displayed a predominantly CD11c+CD8α− splenic DC population and IL-4 intragraft expression characteristic of a Th2 response. In addition, the CD11c+IL-12+ splenic DC population in C57BL/6 recipients was significantly higher than that in BALB/c recipients. Adoptive transfer of syngeneic CD8α− bone marrow-derived DC shifted a Th1-dominant, slow cell-mediated rejection to a Th2-dominant, aggressive acute vascular rejection (AVR) in C57BL/6 mice. This was associated with a cytokine shift from Th1 to Th2 in these mice. In contrast, transfer of CD8α+ bone marrow-derived DC shifted AVR to cell-mediated rejection in BALB/c mice and significantly prolonged graft survival time from 6.0 ± 0.6 days to 14.2 ± 0.8 days. CD8α+ DC transfer rendered BALB/c mice susceptible to cyclosporine therapy, thereby facilitating long-term graft survival. Furthermore, CD8α+ DC transfer in IL-12-deficient mice reconstituted IL-12 expression, induced Th1 response, and attenuated AVR. Our data suggest that the pattern of acute xenogeneic rejection can be regulated by distinct DC subsets.

Adaptive immunity is triggered at the immune synapse, where peptide-major histocompatibility complexes and costimulatory molecules expressed by dendritic cells (DCs) are physically presented to T cells. Here we describe transmission of the inflammatory monoamine serotonin (5-hydroxytryptamine [5-HT]) between these cells. DCs take up 5-HT from the microenvironment and from activated T cells (that synthesize 5-HT) and this uptake is inhibited by the antidepressant, fluoxetine. Expression of 5-HT transporters (SERTs) is regulated by DC maturation, exposure to microbial stimuli, and physical interactions with T cells. Significantly, 5-HT sequestered by DCs is stored within LAMP-1+ vesicles and subsequently released via Ca2+-dependent exocytosis, which was confirmed by amperometric recordings. In turn, extracellular 5-HT can reduce T-cell levels of cAMP, a modulator of T-cell activation. Thus, through the uptake of 5-HT at sites of inflammation, and from activated T cells, DCs may shuttle 5-HT to naive T cells and thereby modulate T-cell proliferation and differentiation. These data constitute the first direct measurement of triggered exocytosis by DCs and reveal a new and rapid type of signaling that may be optimized by the intimate synaptic environment between DCs and T cells. Moreover, these results highlight an important role for 5-HT signaling in immune function and the potential consequences of commonly used drugs that target 5-HT uptake and release.

Interleukin-12 p70 (IL-12p70) is a major dendritic cell (DC)-produced cytokine known to support type-1 T helper (Th1) cells and inflammatory-type immunity. While the ability of DC to produce bioactive IL-12p70 depends on both the DC subtype and the microenvironmental conditions of DC development, the relative contribution of each of these factors remains unclear. Here, we report that in contrast to spleen CD8alpha+ and CD8alpha- DC that show strong differences in their respective IL-12p70-producing capacities, CD8alpha+ and CD8alpha- DC isolated from the liver, a non-lymphoid organ, both efficiently produce IL-12p70 in amounts comparable to spleen CD8alpha+ DC. The IL-12p70-producing capacity CD8alpha+ and CD8alpha- DC from either location is greatly increased following their overnight culture in the presence of granulocyte-macrophage colony-stimulating factor. The elevated production of IL-12p70 by short-term cultured DC correlates with their enhanced expression of CD40 and other costimulatory molecules, and elevated T cell-stimulatory capacity. These data indicate that low IL-12-producing capacity is not an intrinsic property of the CDalpha8- DC subtype, and support the hypothesis that factors such as the site of DC development and maturation stage play a dominant role in defining DC function.

Ca 2+ signaling plays an important role in the function of dendritic cells (DC), the specialized antigen-presenting cells of the immune system. Here we describe functional ryanodine receptor (RyR) Ca 2+ release channels in murine, bone marrow-derived DC. RT-PCR analysis identified selective expression of the type 1 RyR, with higher levels detected in immature rather than mature DC. The RyR activators caffeine, FK506, ryanodine and 4-chloro- m -cresol mobilized Ca 2+ in DC, and responses to 4-chloro- m -cresol were inhibited by dantrolene. Furthermore, activation of RyRs both inhibited subsequent inositol trisphosphate-mediated Ca 2+ release and provoked store-operated Ca 2+ entry, suggesting a functional interaction between these intracellular Ca 2+ channels. Thus, the RyR1 channel may play an intrinsic role in Ca 2+ signaling in DC.

CD8α+ and CD8α− dendritic cells (DCs) arise from committed bone marrow progenitors and can induce or regulate immune reactivity. Previously, the maturational status of CD8α− (myeloid) DCs has been shown to influence allogeneic T cell responses and allograft survival. Although CD8α+ DCs have been implicated in central tolerance and found to modulate peripheral T cell function, their influence on the outcome of organ transplantation has not been examined. Consistent with their equivalent high surface expression of MHC and costimulatory molecules, sorted mature C57BL/10J (B10; H2b) DCs of either subset primed naive, allogeneic C3H/HeJ (C3H; H2k) recipients for Th1 responses. Paradoxically and in contrast to their CD8α− counterparts, mature CD8α+ B10 DCs given systemically 7 days before transplant markedly prolonged B10 heart graft survival in C3H recipients. This effect was associated with specific impairment of ex vivo antidonor T cell proliferative responses, which was not reversed by exogenous IL-2. Further analyses of possible underlying mechanisms indicated that neither immune deviation nor induction of regulatory cells was a significant contributory factor. In contrast to the differential capacity of the mature DC subsets to affect graft outcome, immature CD8α+ and CD8α− DCs administered under the same experimental conditions significantly prolonged transplant survival. These observations demonstrate for the first time the innate capacity of CD8α+ DCs to regulate alloimmune reactivity and transplant survival, independent of their maturation status. Mobilization of such a donor DC subset with capacity to modulate antidonor immunity may have significant implications for the therapy of allograft rejection.

Although Ca2+-signaling processes are thought to underlie many dendritic cell (DC) functions, the Ca2+ entry pathways are unknown. Therefore, we investigated Ca2+-signaling in mouse myeloid DC using Ca2+ imaging and electrophysiological techniques. Neither Ca2+ currents nor changes in intracellular Ca2+ were detected following membrane depolarization, ruling out the presence of functional voltage-dependent Ca2+ channels. ATP, a purinergic receptor ligand, and 1–4 dihydropyridines, previously suggested to activate a plasma membrane Ca2+ channel in human myeloid DC, both elicited Ca2+ rises in murine DC. However, in this study these responses were found to be due to mobilization from intracellular stores rather than by Ca2+ entry. In contrast, Ca2+ influx was activated by depletion of intracellular Ca2+ stores with thapsigargin, or inositol trisphosphate. This Ca2+ influx was enhanced by membrane hyperpolarization, inhibited by SKF 96365, and exhibited a cation permeability similar to the Ca2+ release-activated Ca2+ channel (CRAC) found in T lymphocytes. Furthermore, ATP, a putative DC chemotactic and maturation factor, induced a delayed Ca2+ entry with a voltage dependence similar to CRAC. Moreover, the level of phenotypic DC maturation was correlated with the extracellular Ca2+ concentration and enhanced by thapsigargin treatment. These results suggest that CRAC is a major pathway for Ca2+ entry in mouse myeloid DC and support the proposal that CRAC participates in DC maturation and migration.

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 CD8α. We undertook a comparative study of CD8α+ (CD11blow; lymphoid-related) and CD8α− (CD11bhigh; myeloid) DC isolated from mouse liver. CD8α+ and CD8α− DC each constituted ≤1.0% of the freshly isolated, normal nonparenchymal cells (NPC). Both populations were enriched 10–15% by overnight culture and metrizamide density centrifugation. Flt3 ligand (Flt3L) potently induced equal expansion of both subsets in vivo. Tissue-resident CD8α+ DC, freshly isolated from Flt3L-treated mice, existed primarily as immature cells (CD11c+, CD11blow, CD40−/low, CD80low, CD86low, MHC class IIlow), consistent with previous observations regarding bulk DC freshly isolated from nonlymphoid tissues. Following overnight culture in GM-CSF, CD8α+ DC underwent phenotypic and functional maturation equivalent to that observed for CD8α− DC. CD95 ligand (FasL) mRNA was detected in both immature and mature DC of each subset. In vitro analysis confirmed that flow-sorted, mature CD8α+ and CD8α− DC were strong and equally efficient stimulators of allogeneic T cell proliferation in primary MLR. Both immunohistochemical and genomic DNA analysis revealed that in vivo, sorted CD8α+ DC trafficked from s.c. sites to T cell areas of allogeneic lymphoid tissue and were equally efficient at priming naive T cells compared with CD8α− DC. This is the first comparative study of lymphoid-related DC isolated from nonlymphoid tissue.

Livers transplanted across major histocompatibility complex (MHC) barriers in mice are normally accepted without recipient immune suppression, and induce a state of functional tolerance. However, markedly increasing functional dendritic cells (DC) in the 'passenger leucocyte' population by donor pretreatment with the hematopoietic growth factor Flt3-ligand (Flt3L; 10 microg/day for 10 days) results in acute allograft rejection. In this study, molecular, immunohistochemical and flow cytometric analysis of donor cell traffick into recipient lymphoid tissue 24 h after liver transplantation (C57BL/10 [H2b]--C3H [H2k]) was performed. In addition, the capacity of donor-derived cells in these tissues to stimulate host T cell proliferation was examined. Reverse transcriptase polymerase chain reaction analysis revealed increases in donor genomic DNA in both thymi and spleens of mice given livers from Flt3L-treated donors compared to controls. Donor MHC class II+ (IAb+) cells in spleens were strikingly elevated (10-fold) in the former group. Two-colour flow cytometry revealed a similar increase in donor-derived H-2Kb+/I-Ab+ cells, and in the incidence of donor leucocytes expressing CD40, CD80, and CD86. CD11c+ DC comprised approximately 40% of the I-Ab+ cells in spleens of mice given livers from Flt3L-treated donors. These changes were associated with the presence, in spleens, of potent allostimulatory activity for naive recipient strain T cells, that was not observed in normal liver recipients. Elicitation of allograft rejection, associated with enhanced trafficking of stimulatory donor antigen-presenting cells (APC), in particular DC, suggests that normal liver graft survival and tolerance induction may be linked to failure/counter-regulation of APC-driven stimulation of effective anti-donor T cell responses.

Inflammatory stimuli provide critical activation signals for dendritic cells (DC). Signaling through the capsaicin receptor TRPV1 is reported to initiate DC maturation and migration. We attempted to characterize TRPV1 channels in DC. Capsaicin or extracellular protons failed to elicit a change in intracellular [Ca2+] or membrane current in DC. In contrast, capsaicin evoked a sustained increase in [Ca2+] and large inwards currents in sensory neurons and TRPV1-expressing HEK293 cells. TRPV1 expression was confirmed by RT-PCR in sensory neurons, but was undetectable in DC. Interestingly, and in contrast to capsaicin, the inflammatory neuropeptide substance P evoked Ca2+ transients in DC. Thus, our data do not support the hypothesis that DC express TRPV1 channels. Rather, signaling through TRPV1 in sensory nerves may modulate DC via neurogenic actions.

Tolerance induction by CD45RB monoclonal antibody (mAb) in murine allograft models is associated with an alteration in the CD45RBlo/CD45RBhi T-cell ratio in favor of CD45RBlo T cells, which can function as regulatory cells and promote tolerance. It has been proposed that inversion of the CD45RBhi/CD45RBlo normal T-cell ratio by mAb can occur by down-regulation of CD45RB surface molecules expressed by T cells. Because CD45RB mAb infusion can lead to a reduction in peripheral T cells, we tested whether other mechanisms might participate in the inversion of the CD45RBhi/CD45RBlo ratio, including apoptosis of CD45RBhi cells. We report that CD45RB mAb led to rapid elimination of both CD4+ and CD8+ T cells in vitro. Importantly, CD45RB mAb selectively eliminated CD45RBhi T cells without affecting the viability of CD45RBlo T cells. Furthermore, the death of T cells occurred with a reduction in mitochondrial transmembrane potential and DNA fragmentation but with little evidence of nuclear condensation and cell shrinkage typically found with cells undergoing apoptosis. We propose that CD45RB mAb therapy may promote a dominant regulatory T-cell population that has the capacity to inhibit rejection by the selective elimination of CD45RBhi effector T cells. This occurs by a process that does not involve the classic morphologic features of apoptosis. Strategies that facilitate an inversion of the CD45RBhi/CD45RBlo T-cell subset ratio may improve the efficacy of CD45RB mAb, and therapeutic measures that prevent deletion of CD45RBhi T cells may need to be avoided to achieve tolerance clinically.

A current goal of vaccine development against human immunodeficiency virus (HIV) is to develop a strategy that stimulates long-lasting memory T-cell responses, and provides immediate cytotoxicity in response to viral challenge. We demonstrate that the viral antiapoptotic molecule M11L promotes cellular immune responses to the HIV envelope protein. Coexpression of M11L in vitro inhibits gp140-mediated apoptosis and increases gp140 expression levels. Mice primed with M11L-pHERO DNA, followed by vCP205 boosting, exhibit significantly greater HIV-specific T-cell responses. Moreover, M11L synergizes with CpG motifs to augment anti-HIV responses and stimulates robust expansion of central memory and effector memory CD8+ T-cells. Inclusion of M11L in a DNA vector increases the magnitude of T-cell responses, and promotes the generation of memory T-cells that provide rapid-responding CTL responses. This vaccine strategy may facilitate the generation of an efficacious vaccine for HIV, and other chronic diseases that require enhanced cell-mediated immunity, including HCV and metastatic cancer.