Rapid and long-term disappearance of CD4+ T lymphocyte responses specific for Anaplasma marginale major surface protein-2 (MSP2) in MSP2 vaccinates following challenge with live A. marginale

In humans and ruminants infected with Anaplasma, the major surface protein 2 (MSP2) is immunodominant. Numerous CD4(+) T cell epitopes in the hypervariable and conserved regions of MSP2 contribute to this immunodominance. Antigenic variation in MSP2 occurs throughout acute and persistent infection, and sequentially emerging variants are thought to be controlled by variant-specific Ab. This study tested the hypothesis that challenge of cattle with Anaplasma marginale expressing MSP2 variants to which the animals had been immunized, would stimulate variant epitope-specific recall CD4(+) T cell and IgG responses and organism clearance. MSP2-specific T lymphocyte responses, determined by IFN-gamma ELISPOT and proliferation assays, were strong before and for 3 wk postchallenge. Surprisingly, these responses became undetectable by the peak of rickettsemia, composed predominantly of organisms expressing the same MSP2 variants used for immunization. Immune responsiveness remained insignificant during subsequent persistent A. marginale infection up to 1 year. The suppressed response was specific for A. marginale, as responses to Clostridium vaccine Ag were consistently observed. CD4(+)CD25(+) T cells and cytokines IL-10 and TGF-beta1 did not increase after challenge. Furthermore, a suppressive effect of nonresponding cells was not observed. Lymphocyte proliferation and viability were lost in vitro in the presence of physiologically relevant numbers of A. marginale organisms. These results suggest that loss of memory T cell responses following A. marginale infection is due to a mechanism other than induction of T regulatory cells, such as peripheral deletion of MSP2-specific T cells.     

Comparison of the functional properties of murine dendritic cells generated in vivo with Flt3 ligand, GM-CSF and Flt3 ligand plus GM-SCF

Flt3 ligand (FL) and granulocyte-macrophage colony-stimulating factor (GM-CSF) are important growth factors for dendritic cells (DC). Substantial numbers of DC can be generated in vivo following the administration of either factor. We sought to extend our knowledge of the functional properties of these cells including their ability to prime na?ve CD8(+) T cells. In addition, we compared the nature of the DC generated in vivo with the single cytokines to those generated with the combination of FL+polyethylene glycol-modified GM-CSF (pGM-CSF). Treatment with FL+pGM-CSF yielded greater numbers of both CD11b(low) and CD11b(high) DC than with either cytokine alone, and these DC were more efficient at antigen (Ag) capture. The FL+pGM-CSF-generated CD11b(low) DC lacked expression of CD8alpha. Following treatment with LPS in vivo, all DC subsets upregulated CD40, CD80, CD86, and MHC class II expression, but surprisingly Ag capture was not downregulated and some DC subsets retained expression of intracellular MHC class II vesicles. Thus, even after activation in vivo with LPS, DC retained Ag capture properties of immature DC, and Ag presentation/costimulation properties of mature DC. Though all DC subsets stimulated CD4(+) T cell proliferation equivalently, FL-generated DC were more efficient at priming Ag-specific CD8(+) cytolytic T cells than DC generated with either pGM-CSF alone or FL+pGM-CSF, and CD11b(high) DC were more efficient at priming CD8(+) T cells than CD11b(low) DC.     

Murine Flt3 ligand expands distinct dendritic cells with both tolerogenic and immunogenic properties

Human Flt3 ligand can expand dendritic cells (DC) and enhance immunogenicity in mice. However, little is known about the effects of murine Flt3 ligand (mFlt3L) on mouse DC development and function. We constructed a vector to transiently overexpress mFlt3L in mice. After a single treatment, up to 44% of splenocytes became CD11c(+) and the total number of DC increased 100-fold. DC expansion effects lasted for >35 days. mFlt3L DC were both phenotypically and functionally distinct. They had increased expression of MHC and costimulatory molecules and expressed elevated levels of B220 and DEC205 but had minimal CD4 staining. mFlt3L DC also had a markedly altered cytokine profile, including lowered secretion of IL-6, IL-10, IFN-gamma, and TNF-alpha, but had a slightly increased capacity to stimulate T cells in vitro. However, in a variety of in vivo models, DC expanded by mFlt3L induced tolerogenic effects on T cells. Adoptive transfer of Ag-pulsed mFlt3L splenic DC to naive mice actually caused faster rates of tumor growth and induced minimal CTL compared with control DC. mFlt3L also failed to protect against tumors in which human Flt3 ligand was protective, but depletion of CD4(+) T cells restored tumor protection. Our findings 1) demonstrate that mFlt3L has distinct effects on DC development, 2) suggest an important role for mFlt3L in generating DC that have tolerogenic effects on T cells, and 3) may have application in immunotherapy in generating massive numbers of DC for an extended duration.     

The adjuvanticity of a mannosylated antigen reveals TLR4 functionality essential for subset specialization and functional maturation of mouse dendritic cells

The evidence that dendritic cell (DC) subsets produce differential cytokines in response to specific TLR stimulation is robust. However, the role of TLR stimulation in Ag presentation and phenotypic maturation among DC subsets is not clear. Through the adjuvanticity of a novel mannosylated Ag, mannosylated dendrimer OVA (MDO), as a pathogen-associated molecular pattern Ag, we characterized the functionality of GM-CSF/IL-4-cultured bone marrow DC and Flt3 ligand (Flt3-L) DC subsets by Ag presentation and maturation assays. It was demonstrated that both bone marrow DCs and Flt3-L DCs bound, processed, and presented MDO effectively. However, while Flt3-L CD24(high) (conventional CD8(+) equivalent) and CD11b(high) (CD8(-) equivalent) DCs were adept at MDO processing by MHC class I and II pathways, respectively, CD45RA(+) plasmacytoid DCs presented MDO poorly to T cells. Successful MDO presentation was largely dependent on competent TLR4 for Ag localization and morphological/phenotypic maturation of DC subsets, despite the indirect interaction of MDO with TLR4. Furthermore, Toll/IL-1 receptor-domain-containing adaptor-inducing IFN-beta, but not MyD88, as a TLR4 signaling modulator was indispensable for MDO-induced DC maturation and Ag presentation. Taken together, our findings suggest that DC subsets differentially respond to a pathogen-associated molecular pattern-associated Ag depending on the intrinsic programming and TLRs expressed. Optimal functionality of DC subsets in Ag presentation necessitates concomitant TLR signaling critical for efficient Ag localization and processing.     

Heart, but not skin, allografts from donors lacking Flt3 ligand exhibit markedly prolonged survival time

Fms-like tyrosine kinase 3 ligand (Flt3L) administration leads to dramatic increases in dendritic cells (DC) in lymphoid and nonlymphoid tissues. Conversely, mice lacking Flt3L (Flt3L(-)/(-)) show severe reductions in both myeloid (CD11c(+)CD8alpha(-)) and lymphoid-related DC (CD11c(+)CD8alpha(+)) in the thymus and secondary lymphoid organs. In this study marked reductions in CD11c(+) interstitial cardiac DC and in dermal, but not epidermal, DC (Langerhans cells) were also observed. CD11c(+) cells that migrated from Flt3L(-/-) skin explants expressed lower surface MHC class II and costimulatory molecules and naive T cell allostimulatory activity than migratory wild-type (wt) C57BL/6 (B6) CD11c(+) cells. We examined the survival of Flt3L(-)/(-) heart or tail skin grafts (H2(b)) in allogeneic wt (BALB/c; H2(d)) recipients. The outcome of transplantation of BALB/c organs into Flt3L(-)/(-) recipients was also determined. Flt3L(-)/(-) mice rejected BALB/c heart or skin grafts with similar kinetics as B6 wt recipients. Trafficking of donor DC into host spleens or draining lymph nodes was markedly reduced after transplantation of Flt3L(-)/(-) heart, but not skin grafts, respectively. Compared with wt hearts, survival of Flt3L(-)/(-) hearts was markedly prolonged in BALB/c recipients (median survival time, 37 and 15 days, respectively; p < 0.001). Skin graft survival was unaffected. Rejection of Flt3L(-/-) hearts was precipitated by infusion of wt donor DC at the time of transplant. Thus, severe depletion of interstitial heart DC resulting from targeted gene disruption prolongs, but does not indefinitely extend, heart survival. Acute rejection of wt grafts in Flt3L(-/-) recipients reflects presumably an intact role of the direct pathway of allorecognition.     

Hepatitis C virus structural proteins impair dendritic cell maturation and inhibit in vivo induction of cellular immune responses

Hepatitis C virus (HCV) chronic infection is characterized by low or undetectable cellular immune responses against HCV antigens. Some studies have suggested that HCV proteins manipulate the immune system by suppressing the specific antiviral T-cell immunity. We have previously reported that the expression of HCV core and E1 proteins (CE1) in dendritic cells (DC) impairs their ability to prime T cells in vitro. We show here that immunization of mice with immature DC transduced with an adenovirus encoding HCV core and E1 antigens (AdCE1) induced lower CD4(+)- and CD8(+)-T-cell responses than immunization with DC transduced with an adenovirus encoding NS3 (AdNS3). However, no differences in the strength of the immune response were detected when animals were immunized with mature DC subsequently transduced with AdCE1 or AdNS3. According to these findings, we observed that the expression of CE1 in DC inhibited the maturation caused by tumor necrosis factor alpha or CD40L but not that induced by lipopolysaccharide. Blockade of DC maturation by CE1 was manifested by a lower expression of maturation surface markers and was associated with a reduced ability of AdCE1-transduced DC to activate CD4(+)- and CD8(+)-T-cell responses in vivo. Our results suggest that HCV CE1 proteins modulate T-cell responses by decreasing the stimulatory ability of DC in vivo via inhibition of their physiological maturation pathways. These findings are relevant for the design of therapeutic vaccination strategies in HCV-infected patients.     

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   

Induction of in vivo functional Db-restricted cytolytic T cell activity against a putative phosphate transport receptor of Mycobacterium tuberculosis

Using plasmid vaccination with DNA encoding the putative phosphate transport receptor PstS-3 from Mycobacterium tuberculosis and 36 overlapping 20-mer peptides spanning the entire PstS-3 sequence, we determined the immunodominant Th1-type CD4(+) T cell epitopes in C57BL/10 mice, as measured by spleen cell IL-2 and IFN-gamma production. Furthermore, a potent IFN-gamma-inducing, D(b)-restricted CD8(+) epitope was identified using MHC class I mutant B6.C-H-2(bm13) mice and intracellular IFN-gamma and whole blood CD8(+) T cell tetramer staining. Using adoptive transfer of CFSE-labeled, peptide-pulsed syngeneic spleen cells from naive animals into DNA vaccinated or M. tuberculosis-infected recipients, we demonstrated a functional in vivo CTL activity against this D(b)-restricted PstS-3 epitope. IFN-gamma ELISPOT responses to this epitope were also detected in tuberculosis-infected mice. The CD4(+) and CD8(+) T cell epitopes defined for PstS-3 were completely specific and not recognized in mice vaccinated with either PstS-1 or PstS-2 DNA. The H-2 haplotype exerted a strong influence on immune reactivity to the PstS-3 Ag, and mice of the H-2(b, p, and f) haplotype produced significant Ab and Th1-type cytokine levels, whereas mice of H-2(d, k, r, s, and q) haplotype were completely unreactive. Low responsiveness against PstS-3 in MHC class II mutant B6.C-H-2(bm12) mice could be overcome by DNA vaccination. IFN-gamma-producing CD8(+) T cells could also be detected against the D(b)-restricted epitope in H-2(p) haplotype mice. These results highlight the potential of DNA vaccination for the induction and characterization of CD4(+) and particularly CD8(+) T cell responses against mycobacterial Ags.     

Hematopoietic growth factors in patients receiving intensive chemotherapy for malignant disorders: studies of granulocyte-colony stimulating factor (G-CSF), granulocyte-macrophage colony stimulating factor (GM-CSF), interleukin-3 (IL-3) and Flt-3 ligand (Flt3L)

The levels of hematopoietic growth factors in patients receiving intensive chemotherapy for malignant disorders were investigated using a variety of approaches. Firstly, serum levels of granulocyte-macrophage colony-stimulating factor (GM-CSF), G-CSF and Flt3-ligand (Flt3L) were examined in acute leukemia patients with treatment-induced cytopenia and complicating bacterial infections. Increased serum levels of both G-CSF and Flt3-ligand (Flt3L) were detected when these patients developed therapy-induced leukopenia, whereas GM-CSF levels were low or undetectable. Development of complicating bacterial infections then increased the serum levels of both G- and GM-CSF, and the Flt3L levels remained high during the infections. Secondly, release of growth factors was characterized for clonogenic T cells that remained in the circulation of acute leukemia patients with chemotherapy-induced cytopenia. CD4(+) and CD8(+) T cells from these patients released high levels of GM-CSF, relatively low levels of IL-3 secretion having been detected, and only a minority of the clones released detectable amounts of Flt3L. Thus, circulating T cells may contribute to the high systemic growth factor levels in cytopenic patients. Thirdly, plasma levels of GM-CSF and interleukin-3 (IL-3) were examined in patients with malignant disorders who received chemotherapy plus G-CSF for stem cell mobilization. Increased levels of GM-CSF and Flt3L were detected both in the patients' plasma and in the stem cell grafts. Despite the increased growth factor levels in neutropenic patients with complicating infections, the occurrence of febrile neutropenia did not have a major impact on normal hematopoietic reconstitution (i.e. duration of treatment-induced neutropenia) after intensive chemotherapy for acute myelogenous leukemia.     

A single intratracheal dose of the growth factor Fms-like tyrosine kinase receptor-3 ligand induces a rapid differential increase of dendritic cells and lymphocyte subsets in lung tissue and bronchoalveolar lavage,resulting in an increased local antibody production

Repetitive doses of the growth factor Fms-like tyrosine kinase receptor-3 ligand (Flt3L) have resulted in increased numbers of dendritic cells (DC) in various organs, and the effect on protective or tolerogeneic responses in the gut wall has been documented in the literature. In this study, for the first time, Flt3L was locally applied in the trachea of rats using a single dose only. A dose-dependent increase not only of DC, but also of T lymphocytes (CD4(+) and CD8(+)), was seen with a maximum on day 3. The effects on the cells in the lung interstitium and the bronchoalveolar space showed some differences. The use of tetanus toxoid as a model Ag applied intratracheally after the local Flt3L stimulation resulted in increased levels of specific IgA and IgG in the lung. Thus, this novel approach of locally stimulating APCs by topical application of a DC growth factor before applying the Ag offers a new vaccination strategy.     

Potent CD4+ T cell responses elicited by a bicistronic HIV-1 DNA vaccine expressing gp120 and GM-CSF.

Virus-specific CD4(+) T cell responses have been shown to play a critical role in controlling HIV-1 replication. Candidate HIV-1 vaccines should therefore elicit potent CD4(+) as well as CD8(+) T cell responses. In this report we investigate the ability of plasmid GM-CSF to augment CD4(+) T cell responses elicited by an HIV-1 gp120 DNA vaccine in mice. Coadministration of a plasmid expressing GM-CSF with the gp120 DNA vaccine led to only a marginal increase in gp120-specific splenocyte CD4(+) T cell responses. However, immunization with a bicistronic plasmid that coexpressed gp120 and GM-CSF under control of a single promoter led to a dramatic augmentation of vaccine-elicited CD4(+) T cell responses, as measured by both cellular proliferation and ELISPOT assays. This augmentation of CD4(+) T cell responses was selective, since vaccine-elicited Ab and CD8(+) T cell responses were not significantly changed by the addition of GM-CSF. A 100-fold lower dose of the gp120/GM-CSF bicistronic DNA vaccine was required to elicit detectable gp120-specific splenocyte proliferative responses compared with the monocistronic gp120 DNA vaccine. Consistent with these findings, i.m. injection of the gp120/GM-CSF bicistronic DNA vaccine evoked a more extensive cellular infiltrate at the site of inoculation than the monocistronic gp120 DNA vaccine. These results demonstrate that bicistronic DNA vaccines containing GM-CSF elicit remarkably potent CD4(+) T cell responses and suggest that optimal Th cell priming requires the precise temporal and spatial codelivery of Ag and GM-CSF.     

GM-CSF is an essential regulator of T cell activation competence in uterine dendritic cells during early pregnancy in mice

Uterine dendritic cells (DCs) are critical for activating the T cell response mediating maternal immune tolerance of the semiallogeneic fetus. GM-CSF (CSF2), a known regulator of DCs, is synthesized by uterine epithelial cells during induction of tolerance in early pregnancy. To investigate the role of GM-CSF in regulating uterine DCs and macrophages, Csf2-null mutant and wild-type mice were evaluated at estrus, and in the periconceptual and peri-implantation periods. Immunohistochemistry showed no effect of GM-CSF deficiency on numbers of uterine CD11c(+) cells and F4/80(+) macrophages at estrus or on days 0.5 and 3.5 postcoitum, but MHC class II(+) and class A scavenger receptor(+) cells were fewer. Flow cytometry revealed reduced CD80 and CD86 expression by uterine CD11c(+) cells and reduced MHC class II in both CD11c(+) and F4/80(+) cells from GM-CSF-deficient mice. CD80 and CD86 were induced in Csf2(-/-) uterine CD11c(+) cells by culture with GM-CSF. Substantially reduced ability to activate both CD4(+) and CD8(+) T cells in vivo was evident after delivery of OVA Ag by mating with Act-mOVA males or transcervical administration of OVA peptides. This study shows that GM-CSF regulates the efficiency with which uterine DCs and macrophages activate T cells, and it is essential for optimal MHC class II- and class I-mediated indirect presentation of reproductive Ags. Insufficient GM-CSF may impair generation of T cell-mediated immune tolerance at the outset of pregnancy and may contribute to the altered DC profile and dysregulated T cell tolerance evident in infertility, miscarriage, and preeclampsia.     

Selective induction of dendritic cells using granulocyte macrophage-colony stimulating factor,but not fms-like tyrosine kinase receptor 3-ligand,activates thyroglobulin-specific CD4+/CD25+ T cells and suppresses experimental autoimmune thyroiditis

Fms-like tyrosine kinase receptor 3-ligand (Flt3-L) and GM-CSF cause expansion of different subsets of dendritic cells and skew the immune response toward predominantly Th1 and Th2 type, respectively. In the present study, we investigated their effects on experimental autoimmune thyroiditis in CBA/J mice. Relative to mouse thyroglobulin (mTg) immunized controls, mTg-immunized mice treated with Flt3-L showed more severe thyroiditis characterized by enhanced lymphocytic infiltration of the thyroid, and IFN-gamma and IL-2 production. In contrast, mice treated with GM-CSF, either before or after immunization with mTg, showed suppressed T cell response to mTg and failed to develop thyroiditis. Lymphocytes from these mice, upon activation with mTg in vitro, produced higher levels of IL-4 and IL-10. Additionally, GM-CSF-treated mice showed an increase in the frequency of CD4(+)/CD25(+) T cells, which suppressed the mTg-specific T cell response. Neutralization of IL-10, but not IL-4, or depletion of CD4(+)/CD25(+) cells resulted in increased mTg-specific in vitro T cell proliferation suggesting that IL-10 produced by the Ag-specific CD4(+)/CD25(+) regulatory T cells might be critical for disease suppression. These results indicate that skewing immune response toward Th2, through selective activation of dendritic cells using GM-CSF, may have therapeutic potential in Th1 dominant autoimmune diseases including Hashimoto's thyroiditis.     

CD154 plays a central role in regulating dendritic cell activation during infections that induce Th1 or Th2 responses

We compared splenic DC activation during infection with either the Th2 response-inducing parasite Schistosoma mansoni or with the Th1 response-inducing parasite Toxoplasma gondii. CD8alpha(+) DC from schistosome-infected mice exhibited a 2- to 3-fold increase in the expression of MHC class II, CD80, and CD40 (but not CD86) compared with DC from uninfected control animals, while CD8alpha(-) DC exhibited a 2- to 3-fold increase in the expression of MHC class II and CD80 and no alteration, compared with DC from uninfected mice, in the expression of CD86 or CD40. Intracellular staining revealed that DC did not produce IL-12 during infection with S. mansoni. In contrast, infection with T. gondii resulted in a more pronounced increase in the expression of activation-associated molecules (MHC class II, CD80, CD86, and CD40) on both CD8alpha(-) and CD8alpha(+) splenic DC and promoted elevated IL-12 production by DC. Analysis of MHC class I and of additional costimulatory molecules (ICOSL, ICAM-1, OX40L, 4-1BBL, and B7-DC) revealed a generally similar pattern, with greater indication of activation in T. gondii-infected mice compared with S. mansoni-infected animals. Strikingly, the activation of DC observed during infection with either parasite was not apparent in DC from infected CD154(-/-) mice, indicating that CD40/CD154 interactions are essential for maintaining DC activation during infection regardless of whether the outcome is a Th1 or a Th2 response. However, the ability of this activation pathway to induce IL-12 production by DC is restrained in S. mansoni-infected, but not T. gondii-infected, mice by Ag-responsive CD11c(-) cells.     

Synergistic action of fms-like tyrosine kinase 3 ligand and CD40 ligand in the induction of dendritic cells and generation of antitumor immunity in vivo.

Daily treatment of mice with fms-like tyrosine kinase 3 ligand (Flt3L) leads to a significant increase in the number of dendritic cells and induces antitumor immunity. Here, we show that Flt3L and CD40 ligand (CD40L) synergize in the generation of immune responses against two poorly immunogenic tumors, leading to complete tumor rejection in a high proportion of mice. Rechallenge of the Flt3L + CD40L-treated mice with the immunizing tumor resulted in complete inhibition of tumor growth, indicating that these animals had developed long-lasting antitumor immunity. In addition, we demonstrate that endogenous CD40L plays a critical role in antitumor immunity, since blockade of CD40-CD40L interactions in vivo prevents the generation of antitumor immunity in therapeutic and vaccination protocols. Dendritic cells generated in mice treated with Flt3L alone or in combination with CD40L were equally potent in stimulating allogeneic T cells and expressed similar levels of MHC class II, CD80, and CD86. However, mice treated with Flt3L + CD40L had significantly more dendritic cells than mice treated with either of the cytokines alone, suggesting that CD40L promotes the proliferation and/or survival of dendritic cells generated by Flt3L treatment. Dendritic cells generated in this manner are likely to be involved in the priming of antitumor immune responses.     

Partial regulatory T cell depletion prior to schistosomiasis vaccination does not enhance the protection

CD4(+)CD25(+) regulatory T cells (Tregs) do not only influence self-antigen specific immune responses, but also dampen the protective effect induced by a number of vaccines. The impact of CD4(+)CD25(+) Tregs on vaccines against schistosomiasis, a neglected tropical disease that is a major public health concern, however, has not been examined. In this study, a DNA vaccine encoding a 26 kDa glutathione S-transferase of Schistosoma japonicum (pVAX1-Sj26GST) was constructed and its potential effects were evaluated by depleting CD25(+) cells prior to pVAX1-Sj26GST immunization. This work shows that removal of CD25(+) cells prior to immunization with the pVAX1-Sj26GST schistosomiasis DNA vaccine significantly increases the proliferation of splenocytes and IgG levels. However, CD25(+) cell-depleted mice immunized with pVAX1-Sj26GST show no improved protection against S. japonicum. Furthermore, depletion of CD25(+) cells causes an increase in both pro-inflammatory cytokines (e.g. IFN-γ, GM-CSF and IL-4) and an anti-inflammatory cytokine (e.g. IL-10), with CD4(+)CD25(-) T cells being one of the major sources of both IFN-γ and IL-10. These findings indicate that partial CD25(+) cell depletion fails to enhance the effectiveness of the schistosome vaccine, possibly due to IL-10 production by CD4(+)CD25(-) T cells, or other cell types, after CD25(+) cell depletion during vaccination.     

Endogenous granulocyte-macrophage colony-stimulating factor overexpression in vivo results in the long-term recruitment of a distinct dendritic cell population with enhanced immunostimulatory function

GM-CSF is critical for dendritic cell (DC) survival and differentiation in vitro. To study its effect on DC development and function in vivo, we used a gene transfer vector to transiently overexpress GM-CSF in mice. We found that up to 24% of splenocytes became CD11c+ and the number of DC increased up to 260-fold to 3 x 10(8) cells. DC numbers remained substantially elevated even 75 days after treatment. The DC population was either CD8alpha+CD4- or CD8alpha-CD4- but not CD8alpha+CD4+ or CD8alpha-CD4+. This differs substantially from subsets recruited in normal or Flt3 ligand-treated mice or using GM-CSF protein injections. GM-CSF-recruited DC secreted extremely high levels of TNF-alpha compared with minimal amounts in DC from normal or Flt3 ligand-treated mice. Recruited DC also produced elevated levels of IL-6 but almost no IFN-gamma. GM-CSF DC had robust immune function compared with controls. They had an increased rate of Ag capture and caused greater allogeneic and Ag-specific T cell stimulation. Furthermore, GM-CSF-recruited DC increased NK cell lytic activity after coculture. The enhanced T cell and NK cell immunostimulation by GM-CSF DC was in part dependent on their secretion of TNF-alpha. Our findings show that GM-CSF can have an important role in DC development and recruitment in vivo and has potential application to immunotherapy in recruiting massive numbers of DC with enhanced ability to activate effector cells.     

The Src-like adaptor protein regulates GM-CSFR signaling and monocytic dendritic cell maturation

GM-CSF is an important cytokine involved in myeloid differentiation and inflammatory processes. Signaling through the GM-CSFR also plays a critical role in the generation of monocyte-derived dendritic cells (DC). In this article, we report that the Src-like adaptor protein (SLAP) functions as a negative regulator of the GM-CSFR. In bone marrow-derived DC (BM-DC) lacking SLAP and the closely related SLAP2, downregulation of GM-CSFRβ is impaired, leading to enhanced phosphorylation of Jak2 and prolonged activation of Akt and Erk1/2 in response to GM-CSF stimulation. Compared with wild-type bone marrow, SLAP/SLAP2(-/-) bone marrow gave rise to similar numbers of CD11c(+) and CD11b(+) DC, but SLAP/SLAP2(-/-) BM-DC failed to acquire high levels of MHC class II, CD80, and CD86, indicating an impairment in maturation. Furthermore, MHC class II expression in SLAP/SLAP2(-/-) BM-DC was rescued by decreasing GM-CSF concentration, suggesting that enhanced GM-CSF signaling mediates the block in maturation. In addition, SLAP/SLAP2(-/-) BM-DC produced less IL-12 and TNF-α in response to LPS compared with controls and failed to stimulate T cells in an MLR. Ag-specific T cell activation assays showed that SLAP/SLAP2(-/-) BM-DC were less robust at inducing IFN-γ secretion by DO11.10 T cells. These results indicated that SLAP-mediated GM-CSFR regulation is important for the generation of functionally mature monocytic DC.