Immunization with HIV-1 Gag protein conjugated to a TLR7/8 agonist results in the generation of HIV-1 Gag-specific Th1 and CD8+ T cell responses

One strategy to induce optimal cellular and humoral immune responses following immunization is to use vaccines or adjuvants that target dendritic cells and B cells. Activation of both cell types can be achieved using specific TLR ligands or agonists directed against their cognate receptor. In this study, we compared the ability of the TLR7/8 agonist R-848, which signals only via TLR7 in mice, with CpG oligodeoxynucleotides for their capacity to induce HIV-1 Gag-specific T cell and Ab responses when used as vaccine adjuvants with HIV-1 Gag protein in mice. Injection of R-848 and CpG oligodeoxynucleotides alone enhanced the innate immune responses in vivo as demonstrated by high serum levels of inflammatory cytokines, including IL-12p70 and IFN-alpha, and increased expression of CD80, CD86, and CD40 on CD11c(+) dendritic cells. By contrast, R-848 was a relatively poor adjuvant for inducing primary Th1 or CD8(+) T cell responses when administered with HIV-1 Gag protein. However, when a TLR7/8 agonist structurally and functionally similar to R-848 was conjugated to HIV-1 Gag protein both Th1 and CD8(+) T cells responses were elicited as determined by intracellular cytokine and tetramer staining. Moreover, within the population of HIV-1 Gag-specific CD8(+) CD62(low) cells, approximately 50% of cells expressed CD127, a marker shown to correlate with the capacity to develop into long-term memory cells. Overall, these data provide evidence that TLR7/8 agonists can be effective vaccine adjuvants for eliciting strong primary immune responses with a viral protein in vivo, provided vaccine delivery is optimized.     

Signaling through toll-like receptor 7/8 induces the differentiation of human bone marrow CD34+ progenitor cells along the myeloid lineage

Toll-like receptors (TLRs) play a key role in pathogen recognition and regulation of the innate and adaptive immune responses. Although TLR expression and signaling have been investigated in blood cells, it is currently unknown whether their bone marrow ancestors express TLRs and respond to their ligands. Here we found that TLRs (e.g. TLR4, TLR7 and TLR8) were expressed by freshly isolated human bone marrow (BM) hematopoietic CD34+ progenitor cells. Incubation of these primitive cells with TLR ligands such as immunostimulatory small interfering RNAs and R848, a specific ligand for TLR7/8, induced cytokine production (e.g. IL1-beta, IL6, IL8, TNF-alpha, GM-CSF). Moreover, TLR7/8 signaling induced the differentiation of BM CD34+ progenitors into cells with the morphology of macrophages and monocytic dendritic precursors characterized by the expression of CD13, CD14 and/or CD11c markers. By contrast, R848 ligand did not induce the expression of glycophorin A, an early marker for erythropoiesis. Collectively, the data indicate for the first time that human BM CD34+ progenitor cells constitutively express functional TLR7/TLR8, whose ligation can induce leukopoiesis without the addition of any exogenous cytokines. Thus, TLR signaling may regulate BM cell development in humans.     

The immune response modifier and Toll-like receptor 7 agonist S-27609 selectively induces IL-12 and TNF-alpha production in CD11c+CD11b+CD8- dendritic cells

IL-12 and TNF-alpha production by dendritic cells (DCs) is a critical step in the initiation of local inflammation and adaptive immune responses. We show in this study that a small molecule immune response modifier that is a Toll-like receptor 7 (TLR7) agonist induces IL-12 and TNF-alpha production from murine CD11c(+)CD11b(+)CD8(-) DCs, a subset not previously known for this activity. Stimulation of these DCs through TLR7 in vivo induces significant cytokine production even 12 h after initial stimulation, as well as migration of the DC into T cell zones of the lymphoid tissue. In contrast, stimulation through TLR4 and TLR9 induced IL-12 production predominantly from CD8(+) DCs, consistent with previously published data. All TLR stimuli induced the increase in surface expression of the activation markers B7-1, B7-2, and class II in both CD8(+) and CD8(-) DCs, demonstrating that CD8(+) DCs do respond to TLR7-mediated stimuli. To date this is the only known stimuli to induce preferential cytokine production from CD8(-) DCs. Given the efficacy of TLR7 agonists as antiviral agents, the data collectively indicate that stimulation of CD8(-) DCs through TLR7 most likely plays a role in the generation of antiviral immune responses.     

Adenosinergic regulation of the expansion and immunosuppressive activity of CD11b+Gr1+ cells

Extracellular adenosine and purine nucleotides are elevated in many pathological situations associated with the expansion of CD11b(+)Gr1(+) myeloid-derived suppressor cells (MDSCs). Therefore, we tested whether adenosinergic pathways play a role in MDSC expansion and functions. We found that A(2B) adenosine receptors on hematopoietic cells play an important role in accumulation of intratumoral CD11b(+)Gr1(high) cells in a mouse Lewis lung carcinoma model in vivo and demonstrated that these receptors promote preferential expansion of the granulocytic CD11b(+)Gr1(high) subset of MDSCs in vitro. Flow cytometry analysis of MDSCs generated from mouse hematopoietic progenitor cells revealed that the CD11b(+)Gr-1(high) subset had the highest levels of CD73 (ecto-5'-nucleotidase) expression (Δmean fluorescence intensity [MFI] of 118.5 ± 16.8), followed by CD11b(+)Gr-1(int) (ΔMFI of 57.9 ± 6.8) and CD11b(+)Gr-1(-/low) (ΔMFI of 12.4 ± 1.0) subsets. Even lower levels of CD73 expression were found on Lewis lung carcinoma tumor cells (ΔMFI of 3.2 ± 0.2). The high levels of CD73 expression in granulocytic CD11b(+)Gr-1(high) cells correlated with high levels of ecto-5'-nucleotidase enzymatic activity. We further demonstrated that the ability of granulocytic MDSCs to suppress CD3/CD28-induced T cell proliferation was significantly facilitated in the presence of the ecto-5'-nucleotidase substrate 5'-AMP. We propose that generation of adenosine by CD73 expressed at high levels on granulocytic MDSCs may promote their expansion and facilitate their immunosuppressive activity.     

CD11b+/Gr-1+ myeloid suppressor cells cause T cell dysfunction after traumatic stress

T cell dysfunction that occurs after surgery or trauma is associated with a poor clinical outcome. We describe that myeloid suppressor cells expressing CD11b(+)/Gr-1(+) markers invade the spleen after traumatic stress and suppress T cell function through the production of arginase 1. We created a consistent model of traumatic stress in C57BL/6 mice to perform this work. A significant number of CD11b(+)/Gr-1(+) cells expressing arginase 1 accumulated in T cell zones around the germinal centers of the white pulp of the spleen within 6 h of trauma and lasted for at least 72 h. Increased arginase activity and arginase 1 expression, along with increased [(3)H]arginine uptake, l-arginine depletion, and l-ornithine accumulation in the culture medium, were observed exclusively in CD11b(+)/Gr-1(+) cells after traumatic stress. Flow cytometry revealed CD11b(+)/Gr-1(+) as a heterogeneous myeloid suppressor cell also expressing low levels of MHC class I and II, CD80, CD86, CD31, and others. When compared with controls, trauma-induced CD11b(+)/Gr-1(+) cells significantly inhibited CD3/CD28-mediated T cell proliferation, TCR zeta-chain expression, and IL-2 production. The suppressive effects by trauma CD11b(+)/Gr-1(+) cells were overcome with the arginase antagonist N-hydroxy-nor-l-arginine or extrasupplementation of medium with l-arginine. Poor Ag-presenting capacity of control and trauma-induced CD11b(+)/Gr-1(+) cells was detected in allogeneic murine leukocyte reaction. This study demonstrates that CD11b(+)/Gr-1(+) cells invade the spleen following traumatic stress and cause T cell dysfunction by an arginase-mediated mechanism, probably that of arginine depletion. Understanding the mechanism of immune suppression by these cells has important clinical implications in the treatment of immune dysfunction after trauma or surgery.     

CD11b+Ly-6C(hi) suppressive monocytes in experimental autoimmune encephalomyelitis

Innate immune cells may regulate adaptive immunity by balancing different lineages of T cells and providing negative costimulation. In addition, CD11b(+)Gr-1(+) myeloid-derived suppressor cells have been described in tumor, parasite infection, and severe trauma models. In this study, we observe that splenic CD11b(+) cells markedly increase after experimental autoimmune encephalomyelitis (EAE) immunization, and they suppress T cell proliferation in vitro. Although >80% of CD11b(+) cells express varying levels of Gr-1, only a small population of CD11b(+)Ly-6C(high) inflammatory monocytes (IMC) can efficiently suppress T cell proliferation and induce T cell apoptosis through the production of NO. IFN-gamma produced by activated T cells is essential to induce IMC suppressive function. EAE immunization increases the frequencies of IMC in the bone marrow, spleen, and blood, but not in the lymph nodes. At the peak of EAE, IMC represent approximately 30% of inflammatory cells in the CNS. IMC express F4/80 and CD93 but not CD31, suggesting that they are immature monocytes. Furthermore, IMC have the plasticity to up-regulate NO synthase 2 or arginase 1 expression upon different cytokine treatments. These findings indicate that CD11b(+)Ly-6C(high) IMC induced during EAE priming are powerful suppressors of activated T cells. Further understanding of suppressive monocytes in autoimmune disease models may have important clinical implications for human autoimmune diseases.     

CX3CR1+ c-kit+ bone marrow cells give rise to CD103+ and CD103- dendritic cells with distinct functional properties

Dendritic cells (DC) represent a rather heterogeneous cell population with regard to morphology, phenotype, and function and, like most cells of the immune system, are subjected to a continuous renewal process. CD103(+) (integrin alpha(E)) DC have been identified as a major mucosal DC subset involved in the induction of tissue-specific homing molecules on T cells, but little is known about progenitors able to replenish this DC subset. Herein we report that lineage (lin)(-)CX(3)CR1(+)c-kit(+) (GFP(+)c-kit(+)) bone marrow cells can differentiate to either CD11c(+)CD103(-) or CD11c(+)CD103(+) DC in vitro and in vivo. Gene expression as well as functional assays reveal distinct phenotypical and functional properties of both subsets generated in vitro. CD103(-) DC exhibit enhanced phagocytosis and respond to LPS stimulation by secreting proinflammatory cytokines, whereas CD103(+) DC express high levels of costimulatory molecules and efficiently induce allogeneic T cell proliferation. Following adoptive transfer of GFP(+)c-kit(+) bone marrow cells to irradiated recipients undergoing allergic lung inflammation, we identified donor-derived CD103(+) DC in lung and the lung-draining bronchial lymph node. Collectively, these data indicate that GFP(+)c-kit(+) cells contribute to the replenishment of CD103(+) DC in lymphoid and nonlymphoid organs.     

Toll-like receptor ligands modulate dendritic cells to augment cytomegalovirus- and HIV-1-specific T cell responses

Optimal Ag targeting and activation of APCs, especially dendritic cells (DCs), are important in vaccine development. In this study, we report the effects of different Toll-like receptor (TLR)-binding compounds to enhance immune responses induced by human APCs, including CD123(+) plasmacytoid DCs (PDCs), CD11c(+) myeloid DCs (MDCs), monocytes, and B cells. PDCs, which express TLR7 and TLR9, responded to imidazoquinolines (imiquimod and R-848) and to CpG oligodeoxynucleotides stimulation, resulting in enhancement in expression of costimulatory molecules and induction of IFN-alpha and IL-12p70. In contrast, MDCs, which express TLR3, TLR4, and TLR7, responded to poly(I:C), LPS, and imidazoquinolines with phenotypic maturation and high production of IL-12 p70 without producing detectable IFN-alpha. Optimally TLR ligand-stimulated PDCs or MDCs exposed to CMV or HIV-1 Ags enhanced autologous CMV- and HIV-1-specific memory T cell responses as measured by effector cytokine production compared with TLR ligand-activated monocytes and B cells or unstimulated PDCs and MDCs. Together, these data show that targeting specific DC subsets using TLR ligands can enhance their ability to activate virus-specific T cells, providing information for the rational design of TLR ligands as adjuvants for vaccines or immune modulating therapy.     

A new subset of CD103+CD8alpha+ dendritic cells in the small intestine expresses TLR3, TLR7, and TLR9 and induces Th1 response and CTL activity

CD103(+) dendritic cells (DCs) are the major conventional DC population in the intestinal lamina propria (LP). Our previous report showed that a small number of cells in the LP could be classified into four subsets based on the difference in CD11c/CD11b expression patterns: CD11c(hi)CD11b(lo) DCs, CD11c(hi)CD11b(hi) DCs, CD11c(int)CD11b(int) macrophages, and CD11c(int)CD11b(hi) eosinophils. The CD11c(hi)CD11b(hi) DCs, which are CD103(+), specifically express TLR5 and induce the differentiation of naive B cells into IgA(+) plasma cells. These DCs also mediate the differentiation of Ag-specific Th17 and Th1 cells in response to flagellin. We found that small intestine CD103(+) DCs of the LP (LPDCs) could be divided into a small subset of CD8α(+) cells and a larger subset of CD8α(-) cells. Flow cytometry analysis revealed that CD103(+)CD8α(+) and CD103(+)CD8α(-) LPDCs were equivalent to CD11c(hi)CD11b(lo) and CD11c(hi)CD11b(hi) subsets, respectively. We analyzed a novel subset of CD8α(+) LPDCs to elucidate their immunological function. CD103(+)CD8α(+) LPDCs expressed TLR3, TLR7, and TLR9 and produced IL-6 and IL-12p40, but not TNF-α, IL-10, or IL-23, following TLR ligand stimulation. CD103(+)CD8α(+) LPDCs did not express the gene encoding retinoic acid-converting enzyme Raldh2 and were not involved in T cell-independent IgA synthesis or Foxp3(+) regulatory T cell induction. Furthermore, CD103(+)CD8α(+) LPDCs induced Ag-specific IgG in serum, a Th1 response, and CTL activity in vivo. Accordingly, CD103(+)CD8α(+) LPDCs exhibit a different function from CD103(+)CD8α(-) LPDCs in active immunity. This is the first analysis, to our knowledge, of CD8α(+) DCs in the LP of the small intestine.     

Myeloid-specific expression of human lysosomal acid lipase corrects malformation and malfunction of myeloid-derived suppressor cells in lal-/- mice

Lysosomal acid lipase (LAL) cleaves cholesteryl esters and triglycerides to generate free fatty acids and cholesterol in lysosomes. LAL deficiency causes expansion of CD11b(+)Gr-1(+) immature myeloid cells, loss of T cells, and impairment of T cell function. To test how myeloid cell LAL controls myelopoiesis and lymphopoiesis, a myeloid-specific doxycycline-inducible transgenic system was used to reintroduce human lysosomal acid lipase (hLAL) expression into LAL gene knockout (lal(-/-)) mice. Expression of hLAL in myeloid cells of lal(-/-) mice reversed abnormal myelopoiesis in the bone marrow starting at the granulocyte-monocyte progenitor stage and reduced systemic expansion of myeloid-derived suppressor cells (MDSCs). Myeloid hLAL expression inhibited reactive oxygen species production and arginase expression in CD11b(+)Gr-1(+) cells of lal(-/-) mice. Structural organization of the thymus and spleen was partially restored in association with reduced infiltration of CD11b(+)Gr-1(+) cells in these mice. In the thymus, reconstitution of myeloid cell LAL restored development of thymocytes at the double-negative DN3 stage. Myeloid cell LAL expression improved the proliferation and function of peripheral T cells. In vitro coculture experiments showed that myeloid hLAL expression in lal(-/-) mice reversed CD11b(+)Gr-1(+) myeloid cell suppression of CD4(+) T cell proliferation, T cell signaling activation, and lymphokine secretion. Blocking stat3 and NF-κB p65 signaling by small-molecule inhibitors in MDSCs achieved a similar effect. Injection of anti-Gr-1 Ab into lal(-/-) mice to deplete MDSCs restored T cell proliferation. These studies demonstrate that LAL in myeloid cells plays a critical role in maintaining normal hematopoietic cell development and balancing immunosuppression and inflammation.     

Unopposed production of granulocyte-macrophage colony-stimulating factor by tumors inhibits CD8+ T cell responses by dysregulating antigen-presenting cell maturation

Tumor cells gene-modified to produce GM-CSF potently stimulate antitumor immune responses, in part, by causing the growth and differentiation of dendritic cells (DC). However, GM-CSF-modified tumor cells must be gamma-irradiated or they will grow progressively, killing the host. We observed that 23 of 75 (31%) human tumor lines and two commonly used mouse tumor lines spontaneously produced GM-CSF. In mice, chronic GM-CSF production by tumors suppressed Ag-specific CD8+ T cell responses. Interestingly, an inhibitory population of adherent CD11b(Mac-1)/Gr-1 double-positive cells caused the observed impairment of CD8+ T cell function upon direct cell-to-cell contact. The inhibitory cells were positive for some markers associated with Ag presenting cells, like F4/80, but were negative for markers associated with fully mature DC like DEC205, B7. 2, and MHC class II. We have previously reported that a similar or identical population of inhibitory "immature" APC was elicited after immunization with powerful recombinant immunogens. We show here that these inhibitory cells can be elicited by the administration of recombinant GM-CSF alone, and, furthermore, that they can be differentiated ex vivo into "mature" APC by the addition of IL-4 and GM-CSF. Thus, tumors may be able to escape from immune detection by producing "unopposed" GM-CSF, thereby disrupting the balance of cytokines needed for the maturation of fully functional DC. Further, CD11b/Gr-1 double-positive cells may function as "inhibitory" APC under the influence of GM-CSF alone.     

Toll-like receptor ligands directly promote activated CD4+ T cell survival

Toll-like receptor (TLR) engagement by pathogen-associated molecular patterns (PAMPs) is an important mechanism for optimal cellular immune responses. APC TLR engagement indirectly enhances activated CD4(+) T cell proliferation, differentiation, and survival by promoting the up-regulation of costimulatory molecules and the secretion of proinflammatory cytokines. However, TLRs are also expressed on CD4(+) T cells, suggesting that PAMPs may also act directly on activated CD4(+) T cells to mediate functional responses. In this study, we show that activated mouse CD4(+) T cells express TLR-3 and TLR-9 but not TLR-2 and TLR-4. Treatment of highly purified activated CD4(+) T cells with the dsRNA synthetic analog poly(I:C) and CpG oligodeoxynucleotides (CpG DNA), respective ligands for TLR-3 and TLR-9, directly enhanced their survival without augmenting proliferation. In contrast, peptidoglycan and LPS, respective ligands for TLR-2 and TLR-4 had no effect. Enhanced survival mediated by either poly(I:C) or CpG DNA required NF-kappaB activation and was associated with Bcl-x(L) up-regulation. However, only CpG DNA, but not poly(I:C)-mediated effects on activated CD4(+) T cells required the TLR/IL-1R domain containing adaptor molecule myeloid differentiation factor 88. Collectively, our results demonstrate that PAMPs can directly promote activated CD4(+) T cell survival, suggesting that TLRs on T cells can directly modulate adaptive immune responses.     

Tumor necrosis factor-alpha increases circulating osteoclast precursor numbers by promoting their proliferation and differentiation in the bone marrow through up-regulation of c-Fms expression

Osteoclasts are essential cells for bone erosion in inflammatory arthritis and are derived from cells in the myeloid lineage. Recently, we reported that tumor necrosis factor-alpha (TNFalpha) increases the blood osteoclast precursor (OCP) numbers in arthritic patients and animals, which are reduced by anti-TNF therapy, implying that circulating OCPs may have an important role in the pathogenesis of erosive arthritis. The aim of this study is to investigate the mechanism by which TNFalpha induces this increase in OCP frequency. We found that TNFalpha stimulated cell division and conversion of CD11b+/Gr-1-/lo/c-Fms- to CD11b+/Gr-1-/lo/c-Fms+ cells, which was not blocked by neutralizing macrophage colony-stimulating factor (M-CSF) antibody. Ex vivo analysis of monocytes demonstrated the following: (i) blood CD11b+/Gr-1-/lo but not CD11b-/Gr-1- cells give rise to osteoclasts when they were cultured with receptor activator NF-kappaB ligand and M-CSF; and (ii) TNF-transgenic mice have a significant increase in blood CD11b+/Gr-1-/lo cells and bone marrow proliferating CD11b+/Gr-1-/lo cells. Administration of TNFalpha to wild type mice induced bone marrow CD11b+/Gr-1-/lo cell proliferation, which was associated with an increase in CD11b+/Gr-1-/lo OCPs in the circulation. Thus, TNFalpha directly stimulates bone marrow OCP genesis by enhancing c-Fms expression. This results in progenitor cell proliferation and differentiation in response to M-CSF, leading to an enlargement of the marrow OCP pool. Increased marrow OCPs subsequently egress to the circulation, forming a basis for elevated OCP frequency. Therefore, the first step of TNF-induced osteoclastogenesis is at the level of OCP genesis in the bone marrow, which represents another layer of regulation to control erosive disease.     

Shaping of adaptive immune responses to soluble proteins by TLR agonists: a role for IFN-alpha/beta

Toll-like receptors (TLR) are believed to play a major role in the recognition of invading organisms, although their ability to shape immune responses is not completely understood. Our aim was to investigate in vivo the effect of different TLR stimuli on the generation of antibody responses and the induction of CD8+ T-cell cross-priming after immunization with soluble protein antigens. While all TLR agonists tested elicited the production of immunomodulatory cytokines, marked differences were observed in their ability to stimulate antigen-specific immune responses. Zymosan, poly(I:C) and CpG DNA, which signal through TLR2/6, 3 and 9, respectively, were found to strongly induce the production of IgG2a antibodies, whereas R-848 (TLR7) and LPS (TLR4) did so much more weakly. In contrast, LPS, poly(I:C) and CpG DNA, but not zymosan, induced functional CD8+ T-cell responses against OVA; peptidoglycan (TLR2/?) and R-848 were also ineffective in stimulating cross-priming. Experiments using IFN-alpha/beta R-deficient mice showed that the induction of cross-priming by LPS and poly(I:C) was abrogated in the absence of IFN-alpha/beta signalling, and induction by CpG DNA was greatly reduced. Overall, our results identify LPS as another TLR agonist that is able to generate functional cross-priming against a soluble protein antigen. In addition, our results demonstrate that the ability of TLR stimuli to initiate CD8+ T-cell responses against soluble protein antigens is largely dependent on the IFN-alpha/beta signalling pathway.     

The importance of myeloid-derived suppressor cells in the regulation of autoimmune effector cells by a chronic contact eczema

Induction of a chronic eczema is a most efficient therapy for alopecia areata (AA). We had noted a reduction in regulatory T cells during AA induction and wondered whether regulatory T cells may become recruited or expanded during repeated skin sensitization or whether additional regulatory cells account for hair regrowth. AA could not be cured by the transfer of CD4(+)CD25(high) lymph node cells from mice repeatedly treated with a contact sensitizer. This obviously is a consequence of a dominance of freshly activated cells as compared with regulatory CD4(+)CD25(+) T cells. Instead, a population of Gr-1(+)CD11b(+) cells was significantly increased in skin and spleen of AA mice repeatedly treated with a contact sensitizer. Gr-1(+)CD11b(+) spleen cells mostly expressed CD31. Expression of several proinflammatory cytokines as well as of the IFN-gamma receptor and the TNF receptor I were increased. Particularly in the skin, Gr-1(+) cells expressed several chemokines and CCR8 at high levels. Gr-1(+)CD11b(+) cells most potently suppressed AA effector cell proliferation in vitro and promoted partial hair regrowth in vivo. When cocultured with CD4(+) or CD8(+) cells from AA mice, the Gr-1(+)CD11b(+) cells secreted high levels of NO. However, possibly due to high level Bcl-2 protein expression in AA T cells, apoptosis induction remained unaltered. Instead, zeta-chain expression was strongly down-regulated, which was accompanied by a decrease in ZAP70 and ERK1/2 phosphorylation. Thus, a chronic eczema supports the expansion and activation of myeloid suppressor cells that, via zeta-chain down-regulation, contribute to autoreactive T cell silencing in vitro and in vivo.     

A novel CD4-CD8alpha+CD205+CD11b- murine spleen dendritic cell line: establishment, characterization and functional analysis in a model of vaccination to toxoplasmosis

Dendritic cells (DCs) play an essential role in the induction of immune responses to pathogen infections. Native DCs are difficult to obtain in large numbers and consequently the vast majority of DCs employed in all experiments are derived from bone marrow progenitors. In an attempt to solve this problem, we have established a novel CD8alpha(+) DC line (H-2(k)) from spleen, which we have named SRDC line, and which is easy to culture in vitro. These cells display similar morphology, phenotype and activity to CD4(-)CD8alpha(+)CD205(+)CD11b(-) DCs purified ex vivo. Toxoplasma gondii antigen was shown to be taken up by these cells and to increase class I and class II major histocompatibility complex (MHC), CD40, CD80 and CD86 surface expression. We report that vaccination with T. gondii antigen-pulsed SRDCs, which synthesize large amounts of interleukin-12, induced protective immune responses against this intracellular pathogen in syngeneic CBA/J mice. This protection was associated with strong cellular and humoral immune responses at systemic and intestinal levels. Spleen and mesenteric lymph node cell proliferations were correlated with a Th1/Th2-type response and a specific SRDC homing to spleen and intestine was observed. The SRDC or CD4(-)CD8alpha(+)CD205(+)CD11b(-) DC line can be expected to be a very useful tool for immunobiology studies of DC.     

Expression and function of Toll-like receptors in eosinophils: activation by Toll-like receptor 7 ligand

We investigated the expression of a panel of Toll-like receptors (TLRs) and their functions in human eosinophils. Eosinophils constitutively expressed TLR1, TLR4, TLR7, TLR9, and TLR10 mRNAs (TLR4 greater than TLR1, TLR7, TLR9, and TLR10 greater than TLR6). In contrast, neutrophils expressed a larger variety of TLR mRNAs (TLR1, TLR2, TLR4, TLR6, TLR8 greater than TLR5, TLR9, and TLR10 greater than TLR7). Although the expression levels in eosinophils were generally less prominent compared with those in neutrophils, eosinophils expressed a higher level of TLR7. Furthermore, among various TLR ligands (S-(2,3-bis(palmitoyloxy)-(2-RS)-propyl)-N-palmitoyl-Cys-Ser-(Lys)(4), poly(I:C), LPS, R-848, and CpG DNA), only R-848, a ligand of TLR7 and TLR8, regulated adhesion molecule (CD11b and L-selectin) expression, prolonged survival, and induced superoxide generation in eosinophils. Stimulation of eosinophils by R-848 led to p38 mitogen-activated protein kinase activation, and SB203580, a p38 mitogen-activated protein kinase inhibitor, almost completely attenuated R-848-induced superoxide generation. Although TLR8 mRNA expression was hardly detectable in freshly isolated eosinophils, mRNA expression of TLR8 as well as TLR7 was exclusively up-regulated by IFN-gamma but not by either IL-4 or IL-5. The up-regulation of the TLRs by IFN-gamma had potentially functional significance: the extent of R-848-induced modulation of adhesion molecule expression was significantly greater in cells treated with IFN-gamma compared with untreated cells. Although the natural ligands for TLR7 and TLR8 have not yet been identified, our results suggest that eosinophil TLR7/8 systems represent a potentially important mechanism of a host-defensive role against viral infection and mechanism linking exacerbation of allergic inflammation and viral infection.     

CD209(+) macrophages mediate host defense against Propionibacterium acnes

Propionibacterium acnes is a major etiological factor of acne, triggering an inflammatory response in part through the activation of TLR2. In this study, we demonstrate that activation of peripheral blood monocytes with P. acnes in vitro induced their differentiation into two distinct innate immune cell subsets, CD209(+) macrophages and CD1b(+) dendritic cells. Furthermore, P. acnes induced expression of mRNA for the cytokines IL-15 and GM-CSF, which differentiate CD209(+) and CD1b(+) cells, respectively. The CD209(+) cells were more effective in uptake of P. acnes, compared with the CD1b(+) cells, and demonstrated a 2-fold greater antimicrobial activity against the phagocytosed bacteria. Although CD1b(+) cells secreted inflammatory cytokines in response to both P. acnes and a TLR2 ligand control, the CD209(+) cells responded only to P. acnes. The addition of all-trans retinoic acid, a commonly used agent for the treatment of acne, directly induced differentiation of monocytes into CD209(+) macrophages and enhanced the P. acnes-mediated differentiation of the CD209(+) subset. Therefore, the differentiation of monocytes into CD209(+) macrophages and CD1b(+) dendritic cells distinctly mediate the innate immune response to P. acnes.