[show abstract][hide abstract] ABSTRACT: Despite recent therapeutic advances, including the introduction of novel cytostatic drugs and therapeutic antibodies, many cancer patients will experience recurrent or metastatic disease. Current treatment options, particularly for those patients with metastatic breast, prostate, or skin cancers, are complex and have limited curative potential. Recent clinical trials, however, have shown that cell-based therapeutic vaccines may be used to generate broad-based, antitumor immune responses. Dendritic cells (DC) have proved to be the most efficacious cellular component for therapeutic vaccines, serving as both the adjuvant and antigen delivery vehicle. At present it is not possible to noninvasively determine the fate of DC-based vaccines after their administration to human subjects. In this study, we demonstrate that in vitro-generated mouse DC can be readily labeled with superparamagnetic iron oxide nanoparticles, Feridex, without altering cell morphology, or their phenotypic and functional maturation. Feridex-labeling enables the detection of DC in vivo after their migration to draining lymph nodes using a 1.5 T clinical magnetic resonance scanner. In addition, we report a semiquantitative approach for analysis of magnetic resonance images and show that the Feridex-induced signal void volume, and fractional signal loss, correlates with the delivery and migration of small numbers of in vitro-generated DC. These findings, together with ongoing preclinical studies, are key to gaining information critical for improving the efficacy of therapeutic vaccines for the treatment cancer, and potentially, chronic infectious diseases.
[show abstract][hide abstract] ABSTRACT: 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.
[show abstract][hide abstract] ABSTRACT: Initially, the idea that neurotransmitters could serve as immunomodulators emerged with the discovery that their release and diffusion from nervous tissue could lead to signaling through lymphocyte cell-surface receptors and the modulation of immune function. It is now evident that neurotransmitters can also be released from leukocytes and act as autocrine or paracrine modulators. Here, we review the data indicating that leukocytes synthesize and release 'neurotransmitters' and we also discuss the diverse effects that these compounds exert in a variety of immune cells. The role of neurotransmitters in immune-related diseases is also reviewed succinctly. Current and future developments in understanding the cross-talk between the immune and nervous systems will probably identify new avenues for treating immune-mediated diseases using agonists or antagonists of neurotransmitter receptors.
Trends in Immunology 10/2007; 28(9):400-7. · 9.49 Impact Factor
[show abstract][hide abstract] ABSTRACT: 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-HT(1B) and 5-HT(2A) receptors. Significantly, exogenous 5-HT induces rapid phosphorylation of extracellular signal-regulated kinase-1 and -2 (ERK1/2) and IkappaBalpha in naive T cells. 5-HT-induced activation of ERK1/2 and NFkappaB is inhibited by preincubation with a specific 5-HT(7) receptor antagonist. Thus, 5-HT signaling via the 5-HT(7) 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.
[show abstract][hide abstract] ABSTRACT: Calcium (Ca(2+)) signaling plays a pivotal role in the function of dendritic cells (DC). The Type 1 ryanodine receptor (RyR), a major intracellular Ca(2+) channel, is highly expressed in immature DC. We therefore investigated whether RyR1 plays a role in DC development and function by studying properties of DC derived from wild-type (WT) and RyR1 null [knockout (KO)] mice. Fetal liver cells from WT and RyR1 KO mice retained full hematopoietic competence. Adoptive transfer of these cells into congenic hosts resulted in the generation of functionally equivalent DC populations. WT and RyR1 KO DC exhibited a similar capacity to mature in response to inflammatory and/or activation stimuli, to endocytose antigen, and to stimulate T cell proliferation. Moreover, the absence of RyR1 did not lead to de novo expression of RyR2 or RyR3. WT and RyR KO DC express all three isoforms of inositol 1,4,5-trisphosphate receptor (IP(3)R), although Type 3 IP(3)R gene transcripts are predominant. Further, IP(3)-mediated Ca(2+) transients proceed normally after inhibition of RyRs with dantrolene. Signaling via IP(3)R may therefore be sufficient to drive essential DC Ca(2+) signaling processes in the absence of RyR expression or function.
Journal of Leukocyte Biology 10/2006; 80(3):651-8. · 4.57 Impact Factor
[show abstract][hide abstract] ABSTRACT: 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(+)CD8alpha(+) 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(+)CD8alpha(-) 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 CD8alpha(-) 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 CD8alpha(+) 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. CD8alpha(+) DC transfer rendered BALB/c mice susceptible to cyclosporine therapy, thereby facilitating long-term graft survival. Furthermore, CD8alpha(+) 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.
The Journal of Immunology 04/2006; 176(6):3525-35. · 5.52 Impact Factor
[show abstract][hide abstract] ABSTRACT: 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.
[show abstract][hide abstract] ABSTRACT: 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 [Ca(2+)] or membrane current in DC. In contrast, capsaicin evoked a sustained increase in [Ca(2+)] 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 Ca(2+) 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.
[show abstract][hide abstract] ABSTRACT: We provide phenotypic and functional evidence of premonocytoid dendritic cells (DCs) and preplasmacytoid DCs in blood and of corresponding DC subsets in secondary lymphoid tissue of rhesus monkeys. Subsets were identified and sorted by 4-color flow cytometry using antihuman monoclonal antibodies cross-reactive with rhesus monkey. To mobilize pre-DC subsets, fms-like tyrosine 3 kinase ligand (Flt3L; 100 microg/kg subcutaneously) was administered for 10 days. Presumptive pre-DC subsets were identified within the lineage- (Lin-) major histocompatibility complex (MHC) class II+ fraction of blood mononuclear cells. Premonocytoid DCs were CD11c+CD123- (interleukin-3Ralpha- [IL-3Ralpha-]). Preplasmacytoid DCs were characterized as CD11c-CD123++ Flt3L increased the CD11c+ pre-DC (7-fold) and CD123++ pre-DC subsets (3-fold) in blood. The freshly isolated CD11c+ pre-DC subset induced modest proliferation of naive allogeneic T cells. After overnight culture with granulocyte macro-phage-colony-stimulating factor (GMCSF) and CD40L, both subsets up-regulated surface costimulatory molecules, and CD11c+ pre-DCs became potent allostimulators. Freshly isolated CD123++ pre-DCs showed typical plasmacytoid morphology and, when cultured with IL-3 and CD40L for 72 hours, developed mature DC morphology. Following stimulation with CD40L, CD11c+ pre-DCs secreted increased levels of IL-12p40. Importantly, herpes simplex virus-stimulated CD123++ pre-DCs, but not CD11c+ pre-DCs, secreted interferon-alpha (IFN-alpha). Corresponding DC subsets were identified by flow analysis and immunohistochemistry in lymph nodes wherein both populations were increased 2- to 3-fold by Flt3L administration. CD123+ pre-DCs produced IFN-alpha in response to in vivo viral infection. Thus, rhesus monkeys exhibit 2 distinct DC precursor populations that closely resemble those of humans. Both are mobilized into blood and lymphoid tissue by Flt3L, offering potential for their further characterization and possible therapeutic application.
[show abstract][hide abstract] ABSTRACT: 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.
[show abstract][hide abstract] ABSTRACT: 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.
European Journal of Immunology 08/2003; 33(7):2007-13. · 4.97 Impact Factor
[show abstract][hide abstract] ABSTRACT: 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.
[show abstract][hide abstract] ABSTRACT: Dendritic cells (DC) are rare, bone marrow-derived antigen-presenting cells that play a critical role in the induction and regulation of immune reactivity. In this article, we review the identification and characterization of liver DC, their ontogenic development, in vivo mobilization and population dynamics. In addition, we discuss the functions of DC isolated from liver tissue or celiac lymph, or propagated in vitro from liver-resident haemopoietic stem/progenitor cells. Evidence concerning the role of DC in viral hepatitis, liver tumours, autoimmune liver diseases, granulomatous inflammation and the outcome of liver transplantation is also discussed.
Immunology and Cell Biology 03/2002; 80(1):65-73. · 3.93 Impact Factor
[show abstract][hide abstract] ABSTRACT: CD8alpha+ and CD8alpha- dendritic cells (DCs) arise from committed bone marrow progenitors and can induce or regulate immune reactivity. Previously, the maturational status of CD8alpha-(myeloid) DCs has been shown to influence allogeneic T cell responses and allograft survival. Although CD8alpha+ 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; H2(b)) DCs of either subset primed naive, allogeneic C3H/HeJ (C3H; H2(k)) recipients for Th1 responses. Paradoxically and in contrast to their CD8alpha- counterparts, mature CD8alpha+ 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 CD8alpha+ and CD8alpha- DCs administered under the same experimental conditions significantly prolonged transplant survival. These observations demonstrate for the first time the innate capacity of CD8alpha+ 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.
The Journal of Immunology 02/2002; 168(1):143-54. · 5.52 Impact Factor
[show abstract][hide abstract] ABSTRACT: Background. Considerable interest has focused recently on murine CD8α- and CD8α+ dendritic cell (DC) subsets, because of their roles in initiating and regulating immune responses. Attention has also centered on their presumed human counterparts, DC1 and DC2, respectively, and their precursors. Identification and quantification of these subsets in the blood may be crucial to understanding and monitoring of their immunologic significance, particularly in humans, where blood may be the only tissue readily or routinely available.
Methods. Leukocytes were isolated from anticoagulated human or mouse (C57BL/10J) blood using conventional procedures. Four-color, rare-event, flow cytometric analysis was used to identify DC1 precursors (pDC1; lineage [lin]- CD4+ CD11c+ HLA-DR+) or DC2 precursors (pDC2; lin- CD4+ CD11c- CD123hi [IL-3Rαhi] HLA-DR+) in normal humans. In mice, CD8α+ (CD11blo, CD11c+) and CD8α- (CD11bhi, CD11c+) DC subsets were identified both in normal animals and after administration of the potent DC growth factor, fms-like tyrosine kinase 3 ligand (Flt3L).
Results. All human subjects examined had discrete populations of pDC1 and pDC2 comprising approximately 0.6% and 0.1% of blood mononuclear cells. CD8α- and CD8α+ DC constituted approximately 0.75% and 0.2%, respectively, of blood mononuclear cells in normal mice, and 12% and 0.5%, respectively, in Flt3L-treated animals. Flt3L administration substantially increased the absolute numbers of circulating CD11c+ DC by approximately 200-fold.
Conclusions. In addition to pDC1 and CD8α- DC, pDC2 and CD8α+ DC can be identified in normal human or mouse blood, respectively. Monitoring and isolation or characterization of these cells may provide novel insights into their functional significance in transplantation and other clinical conditions.
[show abstract][hide abstract] ABSTRACT: Although Ca21-signaling processes are thought to underlie many dendritic cell (DC) functions, the Ca 21 entry pathways are unknown. Therefore, we investigated Ca21-signaling in mouse myeloid DC using Ca21 imaging and electrophysiological tech- niques. Neither Ca21 currents nor changes in intracellular Ca21 were detected following membrane depolarization, ruling out the presence of functional voltage-dependent Ca21 channels. ATP, a purinergic receptor ligand, and 1- 4 dihydropyridines, previously suggested to activate a plasma membrane Ca21 channel in human myeloid DC, both elicited Ca21 rises in murine DC. However, in this study these responses were found to be due to mobilization from intracellular stores rather than by Ca 21 entry. In contrast, Ca21 influx was activated by depletion of intracellular Ca 21 stores with thapsigargin, or inositol trisphosphate. This Ca 21 influx was enhanced by membrane hyperpolarization, inhibited by SKF 96365, and exhibited a cation permeability similar to the Ca21 release-activated Ca21 channel (CRAC) found in T lymphocytes. Furthermore, ATP, a putative DC chemotactic and maturation factor, induced a delayed Ca21 entry with a voltage dependence similar to CRAC. Moreover, the level of phenotypic DC matu- ration was correlated with the extracellular Ca21 concentration and enhanced by thapsigargin treatment. These results suggest that CRAC is a major pathway for Ca21 entry in mouse myeloid DC and support the proposal that CRAC participates in DC
The Journal of Immunology 06/2001; 166(10). · 5.52 Impact Factor
[show abstract][hide abstract] ABSTRACT: Background. Liver grafts are accepted across major histocompatibility barriers in mice without immunosuppressive therapy. Potentially tolerogenic immature donor dendritic cells (DC) may play a key role in this phenomenon, but recovery of purified DC from normal livers for functional analysis is inherently difficult. Administration of in vitro propagated immature donor DC to recipients of different types of allograft can prolong transplant survival. By contrast, marked increases in donor liver DC as the result of Flt3 ligand (FL) administration and the resulting augmentation of allostimulatory activity within host lymphoid tissue, is associated with acute graft rejection. Here, we compared the capacity of in vitro generated normal liver immature DC and FL-treated donor liver DC to induce alloimmune CD4+ T helper (Th) 1/Th2 and CD8+ T cytotoxic (Tc) 1/Tc2 responses, in vitro and in vivo.
Methods. B10 (H2b, IAb) immature liver DC were propagated from normal hepatic nonparenchymal cells in granulocyte macrophage-colony stimulating factor (GM-CSF) for 6-8 days. Freshly isolated DC from livers of FL-treated mice (FL-liver DC) were cultured overnight (o/n) in GM-CSF, and both myeloid (CD11c+ CD8α-) and lymphoid DC (CD11c+ CD8α+) flow-sorted for functional analysis. Proliferative activity and production of interferon (IFN)-γ, interleukin (IL)-4, and IL-10 by naive C3H (H2k, IEk) T cells in response to DC stimulation was assessed by [3H]thymidine incorporation, and by multicolor flow cytometric analysis, respectively, after 3-day mixed leukocyte reactions. To investigate their in vivo trafficking, B10 DC were injected subcutaneously into normal C3H mice. Sections of lymphoid tissue were immunostained for donor MHC class II+ (IAb+) cells, and for IFN-γ, IL-4, and IL-10 production. Donor cells and clusters of specific cytokine-secreting cells were enumerated.
Results. Both in vitro propagated normal liver-derived DC, and freshly isolated bulk FL-liver DC showed an immature phenotype (MHC class IIlo, CD40-, CD80-, and CD86-) and were weak stimulators of naive allogeneic T cells. After o/n incubation in GM-CSF, both CD8α- and CD8α+ FL-liver DC exhibited marked up-regulation of surface MHC class II and costimulatory molecules, and acquired potent stimulatory activity for Th1 (mainly) and Th2 cells. Both in vitro propagated immature DC and o/n-cultured mature FL-liver DC homed in vivo to host lymphoid tissues, but with different kinetics. Whereas the mature allogeneic FL-liver DC induced IFN-γ+ clusters in splenic T-cell areas within 2 days, the IFN-γ response to immature DC was much slower and weaker.
Conclusions. FL-treated donor livers that are rejected acutely contain markedly enhanced numbers of myeloid (CD8α-) and lymphoid (CD8α+) DC, many of which are capable of maturing rapidly into strong inducers of Th1 and Tc1 responses. Substantial differences in quantity, and both the phenotypic and functional characteristics of the DC constituency of donor livers, may contribute significantly toward the distinct outcomes of liver transplant tolerance and rejection.
[show abstract][hide abstract] ABSTRACT: 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 μg/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 H2-Kb+/I-Ab+ cells, and in the incidence of donor leucocytes expressing CD40, CD80, and CD86. CD11c+ DC comprised ∼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.
[show abstract][hide abstract] ABSTRACT: Calcium (Ca2) signaling plays a pivotal role in the function of dendritic cells (DC). The Type 1 ryanodine receptor (RyR), a major intra- cellular Ca2 channel, is highly expressed in im- mature DC. We therefore investigated whether RyR1 plays a role in DC development and function by studying properties of DC derived from wild- type (WT) and RyR null (knockout (KO)) mice. Fetal liver cells from WT and RyR1 KO mice re- tained full hematopoietic competence. Adoptive transfer of these cells into congenic hosts resulted in the generation of functionally equivalent DC populations. WT and RyR1 KO DC exhibited a similar capacity to mature in response to inflam- matory and/or activation stimuli, to endocytose an- tigen, and to stimulate T cell proliferation. More- over, the absence of RyR1 did not lead to de novo expression of RyR2 or RyR3. WT and RyR KO DC express all three isoforms of inositol 1,4,5- trisphosphate receptor (IP3R), although Type 3 IP3R gene transcripts are predominant. Further, IP3-mediated Ca 2 transients proceed normally af- ter inhibition of RyRs with dantrolene. Signaling via IP3R may therefore be sufficient to drive essen- tial DC Ca2 signaling processes in the absence of RyR expression or function. J. Leukoc. Biol. 80: 000-000; 2006.