Impaired monocyte maturation in response to CpG oligodeoxynucleotide is related to viral RNA levels in human immunodeficiency virus disease and is at least partially mediated by deficiencies in alpha/beta interferon responsiveness and production

The biological activity of CpG oligodeoxynucleotide 2216 (ODN2216), a Toll-like receptor 9 agonist, was investigated with monocytes from human immunodeficiency virus (HIV)-negative and HIV-positive (HIV+) donors. Exposure of peripheral blood mononuclear cells to CpG ODN2216 led to decreased expression of the monocyte marker CD14 and increased expression of the dendritic cell marker CD83, as well as increased expression of HLA-DR, CD40, CD80, and CD86 among the monocytes. Several features of the CpG ODN-induced maturation were diminished in monocytes from HIV+ donors, and these deficiencies were related to increased viremia but not to CD4 cell counts. Alpha interferon (IFN-alpha) was implicated as at least a partial mediator of the CpG ODN-induced monocyte maturation. Reduced production of IFN-alpha in response to CpG ODN and reduced frequencies of plasmacytoid dendritic cells, the principal IFN-alpha-producing cell type in peripheral blood, were observed in peripheral blood mononuclear cells from HIV+ donors. These deficiencies also were related to levels of plasma HIV RNA. Responses of monocytes from HIV+ donors to direct stimulation with IFN-alpha also were partially impaired. Thus, reduced production of IFN-alpha and reduced IFN-alpha responsiveness may contribute to diminished functional responses to CpG ODN in HIV disease. Application of CpG ODNs in HIV disease for adjuvant or immunoregulatory purposes may be particularly useful for HIV+ donors without high-level viremia.     

Murine plasmacytoid pre-dendritic cells generated from Flt3 ligand-supplemented bone marrow cultures are immature APCs

The putative counterparts of human plasmacytoid pre-dendritic cells (pDCs) have been described in vivo in mouse models and very recently in an in vitro culture system. In this study, we report that large numbers of bone marrow-derived murine CD11c(+)B220(+) pDCs can be generated with Flt3 ligand (FL) as the sole exogenous differentiation/growth factor and that pDC generation is regulated in vivo by FL because FL-deficient mice showed a major reduction in splenic pDC numbers. We extensively analyzed bone marrow-derived CD11c(+)B220(+) pDCs and described their immature APC phenotype based on MHC class II, activation markers, and chemokine receptor level of expression. CD11c(+)B220(+) pDCs showed a nonoverlapping Toll-like receptor pattern of expression distinct from that of classical CD11c(+)B220(-) dendritic cells and were poor T cell stimulators. Stimulation of CD11c(+)B220(+) pDCs with oligodeoxynucleotides containing certain CpG motifs plus CD40 ligand plus GM-CSF led to increased MHC class II, CD80, CD86, and CD8alpha expression levels, to a switch in chemokine receptor expression that affected their migration, to IFN-alpha and IL-12 secretion, and to the acquisition of priming capacities for both CD4(+) and CD8(+) OVA-specific TCR-transgenic naive T cells. Thus, the in vitro generation of murine pDCs may serve as a useful tool to further investigate pDC biology as well as the potential role of these cells in viral immunity and other settings.     

Interferon-producing cells develop from murine CD31(high)/Ly6C(-) marrow progenitors

In this study, we sorted total bone marrow (BM) into six distinct subsets based on surface expression of CD31 and Ly6C and investigated the capacity of these subsets to acquire characteristics of plasmacytoid dendritic cells (PDCs) after in vitro culture with FMS-like tyrosine kinase 3 ligand (Flt3-L). Cultured CD31(high)/Ly6C(-) cells were the only subset that consistently developed immunophenotypic, functional, and morphologic characteristics of PDCs. Culture of this subset resulted in expression of CD11c, B220, and the PDC-specific marker 440C and secretion of interferon-alpha (IFN-alpha) when stimulated with CPG ODN 2216. Cultured cells displayed the typical plasmacytoid morphology of PDCs with eccentrically located nucleus and mature lymphoid chromatin. Unlike conventional dendritic cells (CDCs) that can be generated from CD31(high)/Ly6C(-), CD31(+)/Ly6C(+), and CD31(-)/Ly6C(high) BM subpopulations, PDCs can only be derived from the CD31(high)/Ly6C(-) subset, the subset that reportedly contains the highest frequency of early and late cobblestone area forming cells (CAFC).     

TLR9- and FcepsilonRI-mediated responses oppose one another in plasmacytoid dendritic cells by down-regulating receptor expression

Plasmacytoid dendritic cells (pDC) express not only TLR9 molecules through which ligation with CpG DNA favors Th1 responses but also possess IgE receptors (FcepsilonRI) implicated in allergen presentation and induction of Th2 responses. This dichotomy prompted an investigation to determine whether TLR9- and IgE receptor-mediated responses oppose one another in pDC by affecting receptor expression and associated functional responses. Results showed that IgE cross-linking reduced TLR9 in pDC and inhibited the capacity of these cells to secrete IFN-alpha when stimulated with the CpG oligodeoxynucleotide (ODN)-2216. In contrast, an approximately 15-fold reduction in FcepsilonRIalpha mRNA and a loss in surface protein were seen in pDC first exposed to TLR9 ligation with ODN-2216. Results indicated that type I IFNs partly mediated this effect, as rIFN-alpha also caused a significant approximately 4-fold reduction in FcepsilonRIalpha mRNA. Finally, this reduction in FcepsilonRIalpha mediated by ODN-2216 correlated with a selective suppression of allergen-induced CD4+ T cell proliferation, but not of responses resulting from tetanus toxoid. Overall, these results imply mechanisms by which specific innate and IgE-dependent immune responses counterregulate one another at the dendritic cell level and may have significant impact on whether an ensuing response is either of Th1 or Th2 in nature.     

Identification and characterization of novel bone marrow myeloid DEC205+Gr-1+ cell subsets that differentially express chemokine and TLRs

Bone marrow-derived immunomodulatory cytokines impart a critical function in the regulation of innate immune responses and hemopoiesis. However, the source of immunomodulatory cytokines in murine bone marrow and the cellular immune mechanisms that control local cytokine secretion remain poorly defined. Herein, we identified a population of resident murine bone marrow myeloid DEC205(+)CD11c(-)B220(-)Gr1(+)CD8alpha(-)CD11b(+) cells that respond to TLR2, TLR4, TLR7, TLR8, and TLR9 agonists as measured by the secretion of proinflammatory and anti-inflammatory cytokines in vitro. Phenotypic and functional analyses revealed that DEC205(+)CD11b(+)Gr-1(+) bone marrow cells consist of heterogeneous populations of myeloid cells that can be divided into two main cell subsets based on chemokine and TLR gene expression profile. The DEC205(+)CD11b(+)Gr-1(low) cell subset expresses high levels of TLR7 and TLR9 and was the predominant source of IL-6, TNF-alpha, and IL-12 p70 production following stimulation with the TLR7 and TLR9 agonists CpG and R848, respectively. In contrast, the DEC205(+)CD11b(+)Gr-1(high) cell subset did not respond to CpG and R848 stimulation, which correlated with their lack of TLR7 and TLR9 expression. Similarly, a differential chemokine receptor expression profile was observed with higher expression of CCR1 and CXCR2 found in the DEC205(+)CD11(+)Gr-1(high) cell subset. Thus, we identified a previously uncharacterized population of resident bone marrow cells that may be implicated in the regulation of local immune responses in the bone marrow.     

Human B cells express functional TRAIL/Apo-2 ligand after CpG-containing oligodeoxynucleotide stimulation

CpG-containing oligodeoxynucleotides (CpG ODN) have broad-ranging immunostimulatory effects, including the generation of antitumor immune responses. Analysis of different CpG ODN have identified two classes: CpG-A ODN, which stimulate high levels of IFN-alpha production from plasmacytoid dendritic cells and weakly activate B cells, and CpG-B ODN, which strongly activate B cells but stimulate low production of IFN-alpha from plasmacytoid dendritic cells. Previously, we observed that CpG-B ODN (2006) induces TRAIL/Apo-2 ligand (Apo-2L)-mediated killing of tumor cells by CD14(+) PBMC. In this study, we extend our investigation of CpG ODN-induced TRAIL/Apo-2L expression and activity in PBMC to include CpG-A ODN. Of the two classes, IFN-alpha production and TRAIL/Apo-2L-mediated killing of tumor cells was greatest with CpG-A ODN. Surprisingly, CD3(+), CD14(+), CD19(+), and CD56(+) PBMC expressed high levels of TRAIL/Apo-2L following CpG-A ODN stimulation. When isolated, the CD19(+) PBMC (B cells) were able to kill tumor cells in a TRAIL/Apo-2L-dependent manner. As with CD14(+) PBMC, CD19(+) sorted B cells were capable of up-regulating TRAIL/Apo-2L expression when stimulated with IFN-alpha alone. Interestingly, agonist anti-CD40 mAb further enhanced the IFN-alpha-induced TRAIL/Apo-2L expression on CD19(+) B cells. These results are the first to demonstrate human B cell-mediated killing of tumor cells in a TRAIL/Apo-2L-dependent fashion.     

Failure of NZB spleen to respond to prethymic bone marrow suppressor cells.

Mouse bone marrow contains theta-negative lymphocytes that can suppress an in vitro plaque response by spleen cells primed in vivo with burro red blood cells (BRBC). These bone marrow cells are radiosensitive and can be induced with thymosin fraction 5 or alpha 1 thymic peptides to express the theta antigen. Enrichment for these suppressor pre-T lymphocytes can be achieved by a one-step density centrifugation, macrophage depletion, or a combination of both procedures. NZB mice, which spontaneously develop an autoimmune disorder, have a suppressor abnormality revealed by this assay system. Upon analysis, they have normal BM pre-T suppressor cells but their spleen cells are refractory to the BM suppressor signal. NZB BM suppressor cells inhibit the response by DBA/2 spleen cells, but DBA/2 BM suppressor cells do not inhibit NZB spleen. This resistance to suppression is a property of the B cell fraction recovered from NZB spleen.     

IL-12p70-dependent Th1 induction by human B cells requires combined activation with CD40 ligand and CpG DNA

The detection of microbial molecules via Toll-like receptors (TLR) in B cells is not well characterized. In this study, we found that both naive and memory B cells lack TLR4 (receptor for LPS) but express TLR9 (receptor for CpG motifs) and produce IL-6, TNF-alpha, and IL-10 upon stimulation with CpG oligonucleotides (ODN), synthetic mimics of microbial DNA. Consistent with the lack of TLR4, purified B cells failed to respond to LPS. Similar to CpG ODN, CD40 ligand (CD40L) alone induced IL-6, TNF-alpha, and IL-10. Production of these cytokines as well as IgM synthesis was synergistically increased when both CpG ODN and CD40L were combined. Unlike IL-6, TNF-alpha, and IL-10, the Th1 cytokine IL-12p70 was detected only when both CpG ODN and CD40L were present, and its induction was independent of B cell receptor cross-linking. CpG ODN did not increase the capacity of CD40L-activated B cells to induce proliferation of naive T cells. However, B cells activated with CpG ODN and CD40L strongly enhanced IFN-gamma production in developing CD4 T cells via IL-12. Together, these results demonstrate that IL-12p70 production in human B cells is under the dual control of microbial stimulation and T cell help. Our findings provide a molecular basis for the potent adjuvant activity of CpG ODN to support humoral immune responses observed in vivo, and for the limited value of LPS.     

CpG oligodeoxynucleotide induction of antiviral effector molecules in sheep

Immunostimulatory CpG oligodeoxynucleotide (ODN) can protect mice against infection by many pathogens but the mechanisms mediating disease protection are not well defined. Furthermore, the mechanisms of CpG ODN induced disease protection in vivo have not been investigated in other species. We investigated the induction of antiviral effector molecules in sheep treated with a class B CpG ODN (2007). Subcutaneous injection of ODN 2007 induced a dose-dependent increase in serum levels of the antiviral effector molecule, 2'5'-A synthetase. Peak levels of enzyme were observed 4 days following ODN injection and enzyme levels remained elevated for the following 3-5 days. Repeated ODN injections induced a more sustained elevation of serum 2'5'-A synthetase activity. Finally, formulation of ODN 2007 in emulsigen increased the level of serum 2'5'-A synthetase activity and this response was CpG-specific. Elevated serum 2'5'-A synthetase activity suggested that CpG ODN acted through the induction of either interferon (IFN)-alpha or IFN-gamma. ODN 2007 did not induce detectable levels of IFN-alpha or IFN-gamma when incubated with peripheral blood mononuclear cells, but both IFN-alpha and IFN-gamma were detected following stimulation of lymph node cells with ODN 2007. CpG ODN induction of 2'5'-A synthetase in vitro correlated with the secretion of both IFN-alpha and IFN-gamma. Furthermore, immunohistochemical staining of skin revealed a marked cellular infiltration at the site of ODN 2007 injection. This cellular infiltration was CpG-specific and consisted of primarily CD172(+) myeloid cells. Many of the cells recruited to the site of ODN 2007 injection expressed IFN-alpha and some IFN-gamma. These observations support the conclusion that localized cell recruitment and activation contribute to CpG ODN induction of antiviral effector molecules, such as interferon and 2'5'-A synthetase.     

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.     

A C-type CpG ODN accelerates wound healing via regulating fibroblasts and immune response

Abolished or delayed wound healing is a serious problem in clinical surgery, therefore, the new therapy for wound healing is needed. Synthetic oligodeoxynucleotides containing one or more CpG motifs (CpG ODN) has been reported to activate the immune system and improves skin wound healing. The aim of the present study was to evaluate the role of a new C-type CpG ODN in wound healing. We found that the CpG ODN promoted cell proliferation and collagen I production in human skin fibroblasts cells. Besides, we also investigated the effect of CpG ODN on the activation of immune cells. The macrophages and plasmacytoid dendritic cells (pDCs) were incubated with CpG ODN. CpG ODN activated macrophage and pDCs via regulating TLR9/MyD88/NF-κB pathway and TLR9/MyD88/IRF7 pathway, respectively. To further evaluate the effect of CpG ODN on wound healing in vivo a wound healing model was established in mice. The results showed that CpG ODN treatment accelerated wound healing in mice. CpG ODN increased cytokines secretion in wound skin and elevated the ratio of CD4 ⁺ and CD8 ⁺ T cells in the spleen. Our results showed that CpG ODN accelerated wound healing, which was partly due to the regulation of fibroblasts and immune response. The findings suggested that the CpG ODN might be a proper medicament for the treatment of wound healing.     

Anti-tumor effects of allogeneic bone marrow transplantation in (NZB X NZW)F1 hybrids with spontaneous lymphosarcoma

Untreated female (NZB X NZW)F1 hybrid mice (B/W F1) were found to develop lymphosarcoma spontaneously as they aged. Tumor incidence was evaluated in B/W F1 mice immunosuppressed with total lymphoid irradiation (TLI) and in TLI-conditioned B/W F1 mice reconstituted with 3 X 10(7) BALB/c bone marrow (BM) cells. BALB/C leads to B/W F1 chimerism (79 to 89% BALB/c-type cells) was confirmed by typing peripheral blood lymphocytes with specific alloantisera and complement by using a microcytotoxicity assay. Chimeras showed no clinical signs of graft-vs-host disease (GVHD). TLI-treated mice seemed to show a slightly accelerated onset of lymphosarcoma as compared with untreated controls, but the difference was not significant (p = 0.08). BALB/c leads to B/W F1 chimeras reconstituted at 1 to 3 mo of age (25 mice) developed no tumors for an observation period of 18 mo after transplantation. In contrast, tumors developed in 24/130 of age-matched controls, and in 13/57 of TLI-treated nonreconstituted age-matched B/W F1 mice. Tumor incidence in BALB/c leads to B/W F1 chimeras transplanted at an older age (9 to 11 mo) was similar to that observed in age-matched TLI-treated B/W F1 mice and age-matched untreated controls. The data suggests that the high naturally occurring incidence of lymphosarcoma could be reversed by reconstituting TLI-treated mice with BM cells (p = 0.027). Thus, allogeneic BM transplantation may exert potent graft-vs-tumor effects (GVT) when tumor susceptible hosts are reconstituted at an early age, whereas GVT is relatively ineffective at an advanced age, which probably correlates with an advanced stage of tumor development. Allogeneic BM transplantation should be additionally explored as a potential clinical tool for eradication of certain solid tumors in adjunct to high-dose radiochemotherapy, inasmuch as GVT seems to be independent of GVHD.     

Interleukin 2 induces interferon alpha/beta production in mouse bone marrow cells

We have previously reported that mouse bone marrow (BM) cells stimulated with alloantigen produce cytotoxic effector T-cell activity and produce interferon (IFN-)alpha/beta. In this report we show evidence suggesting that interleukin 2 (IL-2) may play a role in this IFN-alpha/beta production by alloantigen-stimulated BM cells. Alloantigen-induced IFN production by bone marrow cells was completely inhibited when cultures were supplemented with antisera to IL-2. Cell-free supernatants obtained at 2 days from cultures containing C57BL/6 BM cells and irradiated DBA/2J spleen cells were also shown to contain low levels of IL-2 activity and induced significant IFN production in fresh BM cells. Different IL-2 preparations were tested for their ability to induce IFN-alpha/beta production in mouse BM cells. Mouse BM cells cultured with recombinant human IL-2 or highly purified mouse IL-2 produced high levels of IFN-alpha/beta activity after 2-3 days of culture with significant IFN activity being detected as early as 24 hr of culture. IL-2-induced IFN-alpha/beta production was partially resistant to irradiation. In contrast, irradiated (2000 rad) bone marrow cells failed to produce any IFN when cultured with alloantigen in the absence of IL-2. T-cell-depleted BM cells or BM cells obtained from C57BL/10 nude mice produced high levels of IFN-alpha/beta following stimulation with IL-2. In addition, bone marrow cells depleted of Ia+, Qa 5+, or Asialo GM+1 cells produced IFN in response to IL-2. Thus, neither T cells nor NK cells are required for IL-2-induced IFN-alpha/beta production by BM cells. The action of IL-2 on bone marrow cells to induce IFN production was mediated by the classical IL-2 receptor, since monoclonal antibodies to the IL-2 receptor present on T cells blocked this response and since bone marrow cells depleted of IL-2 receptor-bearing cells failed to produce IFN when cultured with IL-2. These results suggest that non-T cells resident in the BM have receptors for IL-2 and can produce IFN-alpha/beta upon stimulation by IL-2. Since IFN has been shown to affect different aspects of hematopoiesis, the production of IFN by BM cells stimulated by IL-2 may be important in the control of hematopoiesis. In addition, IL-2-induced IFN production may play a role in graft-versus-host disease.     

Impaired naive and memory B-cell responsiveness to TLR9 stimulation in human immunodeficiency virus infection

Toll-like receptor 9 (TLR9) agonists such as unmethylated bacterial CpG DNAs activate B lymphocytes directly, potentially influencing their function and homeostasis. To assess B-cell responsiveness to TLR9 agonists in human immunodeficiency virus (HIV) disease, we examined the ability of naive and memory B cells to proliferation and to increase surface expression of CD80 in response to CpG oligonucleotides (ODN). CpG ODN induced expression of CD80 similarly in B cells from HIV-infected persons and from healthy controls. In contrast, proliferation responses to CpG ODN were markedly impaired in both naive and memory B-cell subsets from HIV-infected persons. Naive B-cell proliferation defects were related to plasma HIV RNA and, among memory B cells, to the frequencies of CD21-negative cells. Importantly, TLR9 mRNA levels were significantly diminished in freshly prepared naive B cells and especially so in memory B cells from HIV-positive viremic donors, suggesting a possible underlying mechanism for the observed functional impairments. Dose-response studies indicated that optimal induction of CD80 expression was achieved with much lower concentrations of CpG ODN than optimal induction of proliferation. We propose that the relatively low threshold of activation that is required for CD80 induction by CpG ODN might explain the preservation of this response in B cells from HIV-infected persons despite diminished TLR9 expression. Impaired responsiveness to TLR9 agonists may contribute to defects in humoral immunity in HIV infection.     

Cutting edge: a role for CD1 in the pathogenesis of lupus in NZB/NZW mice

Since anti-CD1 TCR transgenic T cells can activate syngeneic B cells via CD1 to secrete IgM and IgG and induce lupus in BALB/c mice, we studied the role of CD1 in the pathogenesis of lupus in NZB/NZW mice. Approximately 20% of B cells from the spleens of NZB/NZW mice expressed high levels of CD1 (CD1high B cells). The latter subset spontaneously produced large amounts of IgM anti-dsDNA Abs in vitro that was up to 25-fold higher than that of residual CD1int/low B cells. T cells in the NZB/NZW spleen proliferated vigorously to the CD1-transfected A20 B cell line, but not to the parent line. Treatment of NZB/NZW mice with anti-CD1 mAbs ameliorated the development of lupus. These results suggest that the CD1high B cells and their progeny are a major source of autoantibody production, and activation of B cells via CD1 may play an important role in the pathogenesis of lupus.     

Evidence for the existence of two parallel differentiation pathways in the thymus of MRL lpr/lpr mice.

MRL mice homozygous for the lpr/lpr gene develop a massive lymphadenopathy caused by the accumulation of CD4-CD8-, Thy-1-positive T cells that express B220. This phenotypically unusual T cell population coexists with normal, B220- T cells in lpr/lpr animals. To investigate the origin and differentiation pathway of B220+ T cells, the expression of a panel of developmentally regulated cell surface markers including TCR, CD4, CD8, Thy-1, and B220 was examined. Thymocytes and peripheral T lymphocytes from lpr/lpr mice were analyzed by four-color flow cytometry. The results showed that both B220+ and B220- thymocytes contained all of CD4-CD8-, CD4+CD8+, and CD4 or CD8 single positive T cell subpopulation in the lpr thymus. Expression of the V beta 11 TCR, measured by flow cytometry and reverse polymerase chain reaction, was demonstrated in lpr thymus. However, the number of T cells expressing V beta 11 was greatly reduced in both the B220+ and B220- T cell populations in lymph node, spleen, and liver. Taken together, the data provide evidence for maturation and selection of a distinct population of B220+ T cells in the thymus of MRL lpr/lpr mice.     

Mouse strain differences in plasmacytoid dendritic cell frequency and function revealed by a novel monoclonal antibody

We report in this study the generation of a novel rat mAb that recognizes mouse plasmacytoid dendritic cells (pDC). This Ab, named 120G8, stains a small subset of CD11c(low) spleen cell with high specificity. This population produces high amounts of IFN-alpha upon in vitro viral stimulation. Both ex vivo- and in vitro-derived 120G8(+) cells display a phenotype identical with that of the previously described mouse pDC (B220(high)Ly6C(high)Gr1(low)CD11b(-)CD11c(low)). Mice treated with 120G8 mAb are depleted of B220(high)Ly6C(high)CD11c(low) cells and have a much-reduced ability to produce IFN-alpha in response to in vivo CpG stimulation. The mAb 120G8 stains all and only B220(high)Ly6C(high)CD11c(low) pDC in all lymphoid organs. Immunohistochemical studies performed with this mAb indicate that pDC are located in the T cell area of spleen, lymph nodes, and Peyer's patches. Although the Ag recognized by 120G8 is not yet known, we show that its expression is up-regulated by type I IFN on B cells and DC. Using this mAb in immunofluorescence studies demonstrates strain- and organ-specific differences in the frequency of pDC and other DC subsets. 129Sv mice have a much higher frequency of pDC, together with a lower frequency of conventional CD8alpha(+)CD11c(high) DC, compared with C57BL/6 mice, both in spleen and blood. The higher ability of 129Sv mice to produce IFN-alpha in vivo is related to a higher number of pDC, but also to a higher ability of pDC from 129Sv mice to produce IFN-alpha in vitro in response to viral stimulation.     

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 roles of Toll-like receptor 9, MyD88, and DNA-dependent protein kinase catalytic subunit in the effects of two distinct CpG DNAs on dendritic cell subsets

Oligodeoxynucleotides containing unmethylated CpG motifs (CpG DNAs) can function as powerful immune adjuvants by activating APC. Compared with conventional phosphorothioate-backbone CpG DNAs, another type of CpG DNAs, called an A or D type (A/D-type), possesses higher ability to induce IFN-alpha production. Conventional CpG DNAs can exert their activity through Toll-like receptor 9 (TLR9) signaling, which depends on a cytoplasmic adapter, MyD88. However, it remains unknown how A/D-type CpG DNAs exhibit their immunostimulatory function. In this study we have investigated murine dendritic cell (DC) responses to these two distinct CpG DNAs. Not only splenic, but also in vitro bone marrow-derived, DCs could produce larger amounts of IFN-alpha in response to A/D-type CpG DNAs compared with conventional CpG DNAs. This IFN-alpha production was mainly due to the B220(+) DC subset. On the other hand, the B220(-) DC subset responded similarly to both CpG DNAs in terms of costimulatory molecule up-regulation and IL-12 induction. IFN-alpha, but not IL-12, induction was dependent on type I IFN. However, all activities of both CpG DNAs were abolished in TLR9- and MyD88-, but were retained in DNA-PKcs-deficient DCs. This study demonstrates that the TLR9-MyD88 signaling pathway is essential for all DC responses to both types of CpG DNAs.