Characterization of distinct conventional and plasmacytoid dendritic cell-committed precursors in murine bone marrow

The developmental pathways and differentiation relationship of dendritic cell (DC) subsets remain unclear. We report that murine CD11c(+)MHC II(-) bone marrow cells, which are immediate DC precursors of CD8 alpha(+), CD8 alpha(-), and B220(+) DC in vivo, can be separated into B220(+) and B220(-) DC precursor subpopulations. Purified B220(-) DC precursors expand, and generate exclusively mature CD11c(+)CD11b(+)B220(-) DC in vitro and after adoptive transfer. B220(+) DC precursors, which resemble plasmacytoid pre-DC, have a lower proliferative potential than B220(-) DC precursors and generate both CD11b(-) B220(+) and CD11b(+)B220(-) DC populations. Both DC precursor populations can give rise to CD8 alpha(+) and CD8 alpha(-) DC subtypes. Our findings indicate that CD11c(+)MHC II(-)B220(+) and CD11c(+)MHC II(-)B220(-) bone marrow cells are distinct DC lineage-restricted precursors.     

Differential response of respiratory dendritic cell subsets to influenza virus infection

Dendritic cells (DC) are believed to play an important role in the initiation of innate and adaptive immune responses to infection, including respiratory tract infections, where respiratory DC (RDC) perform this role. In this report, we examined the susceptibilities of isolated murine RDC to influenza virus infection in vitro and the effect of the multiplicity of infection (MOI) on costimulatory ligand upregulation and inflammatory cytokine/chemokine production after infection. We found that the efficiency of influenza virus infection of RDC increased with increasing MOIs. Furthermore, distinct subpopulations of RDC differed in their susceptibilities to influenza virus infection and in the magnitude/tempo of costimulatory ligand expression. Additional characterization of the CD11c-positive (CD11c(+)) RDC revealed that the identifiable subsets of RDC differed in susceptibility to infection, with CD11c(+) CD103(+) DC exhibiting the greatest susceptibility, CD11c(+) CD11b(hi) DC exhibiting intermediate susceptibility, and CD11c(+) B220(+) plasmacytoid DC (pDC) exhibiting the least susceptibility to infection. A companion analysis of the in vivo susceptibilities of these RDC subsets to influenza virus revealed a corresponding infection pattern. The three RDC subsets displayed different patterns of cytokine/chemokine production in response to influenza virus infection in vitro: pDC were the predominant producers of most cytokines examined, while CD103(+) DC and CD11b(hi) DC produced elevated levels of the murine chemokine CXCL1 (KC), interleukin 12p40, and RANTES in response to influenza virus infection. Our results indicate that RDC are targets of influenza virus infection and that distinct RDC subsets differ in their susceptibilities and responses to infection.     

Expanded numbers of circulating myeloid dendritic cells in patent human filarial infection reflect lower CCR1 expression

APC dysfunction has been postulated to mediate some of the parasite-specific T cell unresponsiveness seen in patent filarial infection. We have shown that live microfilariae of Brugia malayi induce caspase-dependent apoptosis in human monocyte-derived dendritic cells (DCs) in vitro. This study addresses whether apoptosis observed in vitro extends to patent filarial infections in humans and is reflected in the number of circulating myeloid DCs (mDCs; CD11c(-)CD123(lo)) in peripheral blood of infected microfilaremic individuals. Utilizing flow cytometry to identify DC subpopulations (mDCs and plasmacytoid DCs [pDCs]) based on expression of CD11c and CD123, we found a significant increase in numbers of circulating mDCs (CD11c(+)CD123(lo)) in filaria-infected individuals compared with uninfected controls from the same filaria-endemic region of Mali. Total numbers of pDCs, monocytes, and lymphocytes did not differ between the two groups. To investigate potential causes of differences in mDC numbers between the two groups, we assessed chemokine receptor expression on mDCs. Our data indicate that filaria-infected individuals had a lower percentage of circulating CCR1(+) mDCs and a higher percentage of circulating CCR5(+) mDCs and pDCs. Finally, live microfilariae of B. malayi were able to downregulate cell-surface expression of CCR1 on monocyte-derived DCs and diminish their calcium flux in response to stimulation by a CCR1 ligand. These findings suggest that microfilaria are capable of altering mDC migration through downregulation of expression of some chemokine receptors and their signaling functions. These observations have major implications for regulation of immune responses to these long-lived parasites.     

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.     

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.     

Conditional expression of murine Flt3 ligand leads to expansion of multiple dendritic cell subsets in peripheral blood and tissues of transgenic mice

The analysis of the development and function of distinct subsets of murine dendritic cells (DC) has been hampered by the limited number of these cells in vivo. To circumvent this limitation we have developed a conditional transgenic mouse model for producing large numbers of DC. We used the tetracycline-inducible system to conditionally express murine Flt3 ligand (FL), a potent hemopoietic growth factor that promotes the differentiation and mobilization of DC. Acute treatment (96 h) of the transgenic animals with the tetracycline analog doxycycline (DOX) promoted an approximately 200-fold increase in serum levels of FL without affecting the number of circulating DC. However, within 1 wk of DOX treatment, the relative number of DC in peripheral blood increased from approximately 8 to approximately 40%. Interestingly, both the levels of FL and the number of DC remained elevated for at least 9 mo with continual DOX treatment. Chronic treatment of the mice with DOX led to dramatic increases in the number of DC in multiple tissues without any apparent pathological consequences. Most DC populations were expanded, including immature and mature DC, myeloid (CD11c(+)CD11b(+)CD8a(-)), lymphoid (CD11c(+)CD11b(-)CD8a(+)), and the recently defined plasmacytoid (pDC) subsets. Finally, transplantation of BM from green fluorescent protein-expressing mice into lethally irradiated transgenic mice followed by subsequent DOX treatment led to expansion of green fluorescent protein-labeled DC. The transgenic mice described here should thus provide a readily available source of multiple DC subsets and should facilitate the analysis of their role in homeostasis and disease.     

Efficient Generation of Plasmacytoid Dendritic Cell from Common Lymphoid Progenitors by Flt3 Ligand

Dendritic cells (DCs), including conventional DCs (cDCs) and plasmacytoid DCs (pDCs) are critical for initiating and controlling the immune response. However, study of DC, particularly pDC, function is hampered by their low frequency in lymphoid organs, and existing methods for in vitro DC generation preferentially favor the production of cDCs over pDCs. Here, we demonstrated that pDCs could be efficiently generated in vitro from common lymphoid progenitors (CLPs) using Flt3 ligand (FL) in three different culture systems, namely feeder-free, BM-feeder and AC-6-feeder. This was in stark contrast to common DC progenitors (CDPs), in which cDCs were prominently generated under the same conditions. Moreover, the efficiency and function of pDCs generated from these three systems varied. While AC-6 system showed the greatest ability to support pDC development from CLPs, BM-feeder system was able to develop pDCs with better functionality. pDCs could also be expanded in vivo using hydrodynamic gene transfer of FL, which was further enhanced by the combined treatment of FL and IFN-α. Interestingly, IFN-α selectively promoted the proliferation of CLPs and not CDPs, which might contribute to enhanced pDC development. Together, we have defined conditions for in vitro and in vivo generation of pDCs, which may be useful for investigating the biology of pDCs.     

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.     

Rat plasmacytoid dendritic cells are an abundant subset of MHC class II+ CD4+CD11b-OX62- and type I IFN-producing cells that exhibit selective expression of Toll-like receptors 7 and 9 and strong responsiveness to CpG

We have identified in the rat a new subset of MHC class II(+) CD4(+)CD3(-)CD11b(-) leukocytes that produce high amounts of type I IFN upon viral stimulation and that appeared homologous to plasmacytoid DC (pDC) previously described in humans and mice. These cells exhibited the following phenotype: CD5(+),CD90(+),CD45R(+),CD45RC(+),CD11c(-),CD161a(+),CD200(+),CD172a(+),CD32(+),CD86(+). Rat pDC did not express the DC-specific marker OX62 and were more abundant in the spleen than the classical CD4(+) and CD4(-) subsets of OX62(+)CD11b(+) DC we previously described that produced very little, if any, type I IFN. Spleen pDC exhibited an undifferentiated morphology and rapidly died in vitro, but showed extensive dendrite formation, survival, maturation, and moderate type I IFN production upon stimulation by oligonucleotides containing type B CpG motifs (CpG ODN). Type A CpG ODN and CD40 ligand induced pDC to produce large amounts of type I IFN, but did not promote maturation. CpG ODN and CD40 ligand, but not influenza virus, induced IL-12p40 and IL-6 secretion. Spleen pDC did not produce IL-12p70, TNF-alpha, IL-1beta, or IL-10 using these stimulation conditions. Correlating with their strong responsiveness to virus and CpG ODN, rat pDC specifically expressed Toll-like receptor 7 and 9 mRNA. Fresh spleen pDC were poor stimulators of allogenic CD4(+) and CD8(+) T cells, but became potent inducers of allogenic T cell proliferation as well as Th1 differentiation after stimulation by type B CpG. Therefore, rat pDC appear very similar to human pDC, indicating that the specific phenotype and functions of pDC have been highly conserved between species.     

A role for IFNgamma in differential superantigen stimulation of conventional versus plasmacytoid DCs

Superantigens (SAgs) are known to play a role in food poisoning, toxic shock syndrome and have been identified as a potential mediator of autoimmunity. Although much is known about the effects of SAgs on T cells, by comparison few studies have investigated how SAgs influence innate immune cells. In particular no study has examined how SAgs affect murine plasmacytoid dendritic cells (pDC). We report that in vivo administration of staphylococcal enterotoxin A (SEA) increased the number of pDCs in secondary lymphoid organs, and induced CD86 and CD40 expression. Similar to SEA activation of conventional DCs (cDCs), pDCs relied on T cells, but not on CD40. Nonetheless, pDCs strictly required IFNgamma for upregulation of CD86 and CD40, but cDCs did not depend upon IFNgamma for activation. Further, even though IFNgamma deficient pDCs were not activated by SEA, they were still capable of producing wild-type levels of IFNalpha in response to CpG oligodeoxynucleotide (ODN). The source of IFNgamma for pDC activation was not T cells, nor did pDCs themselves have to synthesize or bind IFNgamma, but the presence of IFNgamma was essential. After SEA stimulation, IFNgamma deficient mice fail to induce expression of the pDC dependent chemokines CXCL9, and demonstrated a defect in recruitment of pDCs to marginal zones of lymphoid organs. Thus, SEA exerts its combined effect on pDC activation, recruitment and chemokine induction through the action of IFNgamma. This fundamental dichotomy of the effects of SAgs on pDCs versus cDCs show how a non-PAMP from bacteria, can selectively and indirectly stimulate innate cell subpopulations much in the same way that differential TLR expression influences cells of the innate immune system.     

Development of murine plasmacytoid dendritic cells defined by increased expression of an inhibitory NK receptor, Ly49Q

Plasmacytoid dendritic cells (PDCs) are defined in mice by a unique combination of markers: CD11c, B220, and Ly6C/G. We have reported previously that PDCs express Ly49Q, a lectin-type killer cell inhibitory receptor. We now find that different expression levels of Ly49Q define sequential developmental stages of PDCs in bone marrow. Although PDCs in spleen and lymph nodes express high levels of Ly49Q, a significant portion of CD11c(+)B220(+) PDCs in bone marrow lack Ly49Q, as well as the CD4 and MHC II. Purified Ly49Q(-) marrow PDCs spontaneously up-regulate Ly49Q after overnight culture without cell proliferation and acquire most features of typical PDCs in spleen. When exposed to TLR ligands, such as CpG-oligodeoxynucleotide and hemagglutinating virus of Japan (Sendai virus), Ly49Q(-) PDCs increase CD86 and MHC class II expression but produce less IFN-alphabeta, IL-6, and IL-12p70 than Ly49Q(+) PDCs, although they are able to produce comparable amounts of TNF-alpha. However, interestingly, Ly49Q(-) PDCs do not produce TNF-alpha in response to the TLR2 ligand, Pam3SCK(4), whereas Ly49Q(+) PDCs did. Therefore, Ly49Q is a new marker to identify a precursor form of PDCs that participates in innate immunity.     

Identification and characterization of pDC-like cells in normal mouse skin and melanomas treated with imiquimod

Among the different subsets of dendritic cells (DC) described in humans and mice, epidermal Langerhans cells and dermal DCs represent the only DC populations resident in normal skin. In this study we describe a population of CD4(+)CD3(-) plasmacytoid DC (pDC)-like cells that accumulate in the dermis and spleens of mice topically treated with imiquimod, a low m.w. immune response modifier with potent antiviral and antitumor activities. These CD4(+)CD3(-) cells coexpress GR-1, B220, MHC class II, and, to a lesser extent, CD11c and display the phenotypic features of pDCs described in lymphoid organs. The accumulation of pDC-like cells after imiquimod treatment was detected not only in normal skin, but also in intradermally induced melanomas. Imiquimod treatment leads either to complete regression or to a significant reduction of the tumors. The number of pDCs correlates well with the clinical response of the tumors to the drug, suggesting that the antitumor effects of imiquimod could be mediated at least in part by the recruitment of pDC-like cells to the skin. Therefore, strategies aimed at activating and directing these cells into neoplastic tissues may be a promising and novel approach for the immunotherapy of various types of cancer.     

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.     

Modulation of haematopoietic progenitor development by FLT-3 ligand.

The Flt-3 receptor is expressed in primitive haematopoietic cells and its ligand exerts proliferative effects on these cells in vitro in synergy with other cytokines. To increase our knowledge of the functional properties of the human Flt-3 ligand (FL) as relating to in vitro expansion of haematopoietic stem cells, the effects on murine haematopoiesis of FL alone or in combination with other growth factors were studied. Analysis of Flk-2/Flt-3 mRNA expression indicated that Flk-2/Flt-3 was preferentially expressed in primitive haematopoietic cell populations. To examine the expression of the Flk-2/Flt-3 receptor on megakaryocyte progenitors (CFU-Meg), Flk-2/Flt-3 positive and negative CD34(+)populations were separated from human bone marrow and cultured in a plasma clot culture system. CFU-Meg colonies were found in the Flk-2/Flt-3 negative fraction. Myeloid (CFU-GM) derived colonies appeared in the presence of FL alone. Neither FL+IL-3 nor FL+IL-3+IL-6 had any effect on the generation of megakaryocyte colonies (CFU-MK), due to the lack of FL receptor expression on megakaryocyte progenitors. Bone marrow cells remaining after 5-fluorouracil (5-FU) treatment of mice represent a very primitive population of progenitors enriched for reconstituting stem cells. This cell population expressed FL receptors, as revealed by RT-PCR analysis. Addition of FL alone did not enhance the replication of such cells in liquid cultures as compared to controls. However, a significantly greater generation of myeloid progenitors (CFU-GM) in clonogenic assays was observed in the presence of FL+IL-3, FL+GM-CSF or FL+CSF-1. In addition, the effects of FL on in vitro expansion of murine haematopoietic stem cells were studied using lineage-negative (lin(-)) Sca-1 positive (Sca-1(+)) c-kit positive (c-kit(+)) marrow cells from 5-FU treated mice. FL enhanced the survival of primitive murine lin(-)Sca-1(+)c-kit(+)cells. FL and IL-6 were able to significantly expand murine progenitor stem cells in vitro and promote their survival. These studies strongly suggest that FL significantly and selectively enhanced the generation of myeloid progenitors in vitro and increased myeloid progenitor responsiveness to later acting growth factors. In addition, FL synergized with IL-6 to support in vitro expansion of haematopoietic progenitors and promoted the survival of lin(-)Sca-1(+)c-kit(+)cells.     

Bone marrow-derived IFN-producing killer dendritic cells account for the tumoricidal activity of unpulsed dendritic cells

The CD11c(int)B220(+)NK1.1(+)CD49(+) subset of cells has recently been described as IFN-producing killer dendritic cells (IKDC), which share phenotypic and functional properties of dendritic cells and NK cells. Herein we show that bone marrow-derived murine dendritic cell preparations contain abundant CD11c(int)B220(+)NK1.1(+)CD49(+) cells, the removal of which results in loss of tumoricidal activity of unpulsed dendritic cells in vivo. Moreover, following s.c. injection, as few as 5 x 10(3) highly pure bone marrow-derived IKDC cells are capable of shrinking small contralateral syngeneic tumors in C57BL/6 mice, but not in immunodeficient mice, implying the obligate involvement of host effector cells in tumor rejection. Our data suggest that bone marrow-derived IKDC represent a population that has powerful tumoricidal activity in vivo.     

The countervailing actions of myeloid and plasmacytoid dendritic cells control autoimmune diabetes in the nonobese diabetic mouse

Islet Ag-specific CD4(+) T cells receive antigenic stimulation from MHC class II-expressing APCs. Herein, we delineate the direct in vivo necessity for distinct subsets of macrophages and dendritic cells (DC) in type 1 diabetes mellitus of the NOD mouse by using diphtheria toxin-mediated cell ablation. The ablation of macrophages had no impact on islet Ag presentation or on the induction of insulitis or diabetes in either transfer or spontaneous models. However, the ablation of CD11b(+)CD11c(+) DC led to the loss of T cell activation, insulitis, and diabetes mediated by CD4(+) T cells. When the specific myeloid DC subset was "added-back" to mice lacking total DC, insulitis and diabetes were restored. Interestingly, when NOD mice were allowed to progress to the insulitis phase, the ablation of DC led to accelerated insulitis. This accelerated insulitis was mediated by the loss of plasmacytoid DC (pDC). When pDC were returned to depleted mice, the localized regulation of insulitis was restored. The loss of pDC in the pancreas itself was accompanied by the localized loss of IDO and the acceleration of insulitis. Thus, CD11c(+)CD11b(+) DC and pDC have countervailing actions in NOD diabetes, with myeloid DC providing critical antigenic stimulation to naive CD4(+) T cells and pDC providing regulatory control of CD4(+) T cell function in the target tissue.     

An NKp30-Based Chimeric Antigen Receptor Promotes T Cell Effector Functions and Antitumor Efficacy In Vivo

NKp30 is a natural cytotoxicity receptor that is expressed on NK cells and recognizes B7-H6, which is expressed on several types of tumors but few normal cells. To target effector T cells against B7-H6(+) tumors, we developed several chimeric AgRs (CARs) based on NKp30, which contain the CD28- and/or CD3ζ-signaling domains with the transmembrane domains from CD3ζ, CD28, or CD8α. The data show that chimeric NKp30-expressing T cells responded to B7-H6(+) tumor cells. The NKp30 CAR-expressing T cells produced IFN-γ and killed B7-H6 ligand-expressing tumor cells; this response was dependent upon ligand expression on target cells but not on MHC expression. PBMC-derived dendritic cells also express NKp30 ligands, including immature dendritic cells, and they can stimulate NKp30 CAR-bearing T cells to produce IFN-γ, but to a lesser extent. The addition of a CD28-signaling domain significantly enhanced the activity of the NKp30 CAR in a PI3K-dependent manner. Adoptive transfer of T cells expressing a chimeric NKp30 receptor containing a CD28-signaling domain inhibited the growth of a B7-H6-expressing murine lymphoma (RMA/B7-H6) in vivo. Moreover, mice that remained tumor-free were resistant to a subsequent challenge with the wild-type RMA tumor cells, suggesting the generation of immunity against other tumor Ags. Overall, this study demonstrates the specificity and therapeutic potential of adoptive immunotherapy with NKp30 CAR-expressing T cells against B7-H6(+) tumor cells in vivo.     

DAP12 signaling regulates plasmacytoid dendritic cell homeostasis and down-modulates their function during viral infection

DAP12 is an ITAM-containing adaptor molecule conveying activating properties to surface receptors on many cell types. We show here that DAP12 paradoxically down-modulates plasmacytoid dendritic cell (pDC) cytokine production in vivo during murine CMV (MCMV) infection. Higher levels of IFN-alphabeta and IL-12 were detected upon MCMV infection or CpG treatment in DAP12-deficient (DAP12(o)) mice as compared with wild-type (WT) mice. This resulted from altered homeostasis and enhanced responsiveness of pDCs in DAP12(o) animals. Increased numbers of pDCs were observed in the periphery of both naive and MCMV-infected DAP12(o) mice. A higher proportion of pDCs was activated in infected DAP12(o) mice, as demonstrated by intracellular staining using an optimized protocol for simultaneous detection of IFN-alpha and IFN-beta. The homeostasis of WT and DAP12(o) pDCs did not differ in mixed bone marrow chimeric mice. In addition, a similar efficiency of pDC differentiation was observed in vitro in Fms-like tyrosine kinase receptor 3 ligand cultures of WT and DAP12(o) bone marrow cells. This suggests that DAP12 signaling effects on pDC homeostasis are indirect. In contrast, in response to CpG, DAP12-mediated effects on both IL-12 and IFN-alphabeta production were intrinsic to the pDCs. However, in response to MCMV, only IL-12 but not IFN-alphabeta production was affected by pDC-intrinsic DAP12 signaling. Thus, DAP12 signaling in pDCs can mediate different regulatory effects on their functions, depending on the mechanisms of pDC activation. The potential implications of the regulation of pDC functions by DAP12 for promoting health over disease are discussed.