Redirecting dendritic cells and macrophages towards tumor rejection

The liver expressed chemokine CCL16/LEC exerts chemotactic activity on human monocytes and lymphocytes and is also active on murine cells. An adenovirus encoding the chemokine CCL16 was used to test whether this chemokine might inhibit pre-existing tumors in mice. AdCCL16, but not control empty vector, when injected in established TSA mammary carcinoma, significantly delayed tumor growth. Immunohistochemistry revealed accumulation at tumor site of CD4+ and CD8+ T cells, macrophages, and dendritic cells (DC) the latter being also enriched in draining lymph nodes. Despite the robust and rapid immune response triggered by intratumoral injection of AdCCL16, the lesions were not completely rejected. However the same treatment given before surgical excision of primary lesions prevented metastatic spread and cured 63% of mice bearing the 4T1 mammary adenocarcinoma. The finding of an hostile tumor microenvironment preventing innate immunity explains the weak effect on the primary tumor. As CCL16 promotes accumulation of macrophages and DC at the site of pre-established tumor nodules this treatment was combined with the TLR9 ligand CpG and with anti-interleukin 10-receptor (IL10R) antibody to contrast the local tumor-induced immunosuppression. CpG plus anti- IL10R promptly switched M2 infiltrating macrophages to Ml that, triggering a strong innate response, debulked large tumors within 16 hrs. Tumor infiltrating DC matured and migrated in parallel with the onset of the innate response, allowing the initiation of adaptive immunity before the diffuse hemorrhagic necrosis halted the communication between tumor and draining lymph node. Treatment of B6>CXB6 chimeras implanted with BALB/c tumors induced an efficient innate response but not CTL-mediated tumor lysis; in these mice tumor rejection did not exceed 25%. The requirement of CD4 help for an effective CTL induction was shown in CD40KO, as well as in mice depleted for CD4 T cells during the priming rather than the effector phase. Together the data describe the critical requirements for the immunological rejection of large tumors: a hemorrhagic necrosis initiated by activated Ml macrophages and a concomitant DC-migration to DLN for subsequent CTL priming and clearing of any tumor remnants

Leukocyte, Rather than Tumor-produced SPARC, Determines Stroma and Collagen Type IV Deposition in Mammary Carcinoma

Secreted protein, acidic and rich in cysteine (SPARC), also known as osteonectin or BM-40, is a Ca2+-binding matricellular glycoprotein involved in development, wound healing, and neoplasia. However, the role of SPARC in tumors is ill defined mostly because it is expressed by both tumor and stromal cells, especially inflammatory cells. We analyzed the respective roles of host- and tumor-derived SPARC in wild-type and congenic SPARC knockout (SPARC−/−) mice on a BALB/c genetic background injected into the mammary fat pad with SPARC-producing mammary carcinoma cells derived from c-erB2 transgenic BALB/c mice. Reduced tumor growth but massive parenchyma infiltration, with large areas of necrosis and impaired vascularization were observed in SPARC−/− mice. Immunohistochemical analysis showed a defect in collagen type IV deposition in the stroma of lobular tumors from SPARC−/− mice. Chimeric mice expressing SPARC only in bone marrow–derived cells were able to organize peritumoral and perilobular stroma, whereas reciprocal chimeras transplanted with bone marrow from SPARC−/− mice developed tumors with less defined lobular structures, lacking assembled collagen type IV and with a parenchyma heavily infiltrated by leukocytes. Together, the data indicate that SPARC produced by host leukocytes, rather than the tumor, determines the assembly and function of tumor-associated stroma through the organization of collagen type IV

PI3K is critical for the nuclear translocation of IRF-7 and type I IFN production by human plasmacytoid predendritic cells in response to TLR activation

Plasmacytoid predendritic cells (pDCs) are the main producers of type I interferon (IFN) in response to Toll-like receptor (TLR) stimulation. Phosphatidylinositol-3 kinase (PI3K) has been shown to be activated by TLR triggering in multiple cell types; however, its role in pDC function is not known. We show that PI3K is activated by TLR stimulation in primary human pDCs and demonstrate, using specific inhibitors, that PI3K is required for type I IFN production by pDCs, both at the transcriptional and protein levels. Importantly, PI3K was not involved in other proinflammatory responses of pDCs, including tumor necrosis factor α and interleukin 6 production and DC differentiation. pDCs preferentially expressed the PI3K δ subunit, which was specifically involved in the control of type I IFN production. Although uptake and endosomal trafficking of TLR ligands were not affected in the presence of PI3K inhibitors, there was a dramatic defect in the nuclear translocation of IFN regulatory factor (IRF) 7, whereas nuclear factor κB activation was preserved. Thus, PI3K selectively controls type I IFN production by regulating IRF-7 nuclear translocation in human pDCs and could serve as a novel target to inhibit pathogenic type I IFN in autoimmune diseases

Triggering CD40 on endothelial cells contributes to tumor growth

Inflammatory cells can either promote or inhibit tumor growth. Here we studied whether CD40, a key molecule for adaptive immune response, has any role in mammary carcinogenesis of BALB/NeuT transgenic tumor-prone mice. We transferred the HER2/neu oncogene into CD40-null background to obtain the CD40-KO/NeuT strain. CD40-KO/NeuT mice showed delayed tumor onset and reduced tumor multiplicity. BM (BM) transplantation experiments excluded a role of BM-derived cells in the reduced tumorigenicity associated with CD40 deficiency. Rather, CD40 expressed by endothelial cells (ECs) takes part to the angiogenic process. Accordingly, large vessels, well organized around the tumor lobular structures, characterize BALB/NeuT tumors, whereas tiny numerous vessels with scarce extracellular matrix are dispersed in the parenchyma of poorly organized CD40-KO/NeuT tumors

Properties regulating the nature of the plasmacytoid dendritic cell response to Toll-like receptor 9 activation

Human plasmacytoid dendritic cells (PDCs) can produce interferon (IFN)-α and/or mature and participate in the adaptive immune response. Three classes of CpG oligonucleotide ligands for Toll-like receptor (TLR)9 can be distinguished by different sequence motifs and different abilities to stimulate IFN-α production and maturation of PDCs. We show that the nature of the PDC response is determined by the higher order structure and endosomal location of the CpG oligonucleotide. Activation of TLR9 by the multimeric CpG-A occurs in transferrin receptor (TfR)-positive endosomes and leads exclusively to IFN-α production, whereas monomeric CpG-B oligonucleotides localize to lysosome-associated membrane protein (LAMP)-1–positive endosomes and promote maturation of PDCs. However, CpG-B, when complexed into microparticles, localizes in TfR-positive endosomes and induces IFN-α from PDCs, whereas monomeric forms of CpG-A localize to LAMP-1–positive endosomes accompanied by the loss of IFN-α production and a gain in PDC maturation activity. CpG-C sequences, which induce both IFN-α and maturation of PDCs, are distributed in both type of endosomes. Encapsulation of CpG-C in liposomes stable above pH 5.75 completely abrogated the IFN-α response while increasing PDC maturation. This establishes that the primary determinant of TLR9 signaling is not valency but endosomal location and demonstrates a strict compartmentalization of the biological response to TLR9 activation in PDCs

RNA recognition by human TLR8 can lead to autoimmune inflammation.

Studies on the role of the RNA receptor TLR8 in inflammation have been limited by its different function in human versus rodents. We have generated multiple lines of transgenic mice expressing different levels of human TLR8. The high copy number chimeras were unable to pass germline; developed severe inflammation targeting the pancreas, salivary glands, and joints; and the severity of the specific phenotypes closely correlated with the huTLR8 expression levels. Mice with relatively low expression levels survived and bred successfully but had increased susceptibility to collagen-induced arthritis, and the levels of huTLR8 correlated with proinflammatory cytokines in the joints of the animals. At the cellular level, huTLR8 signaling exerted a DC-intrinsic effect leading to up-regulation of co-stimulatory molecules and subsequent T cell activation. A pathogenic role for TLR8 in human diseases was suggested by its increased expression in patients with systemic arthritis and the correlation of TLR8 expression with the elevation of IL-1β levels and disease status. We found that the consequence of self-recognition via TLR8 results in a constellation of diseases, strikingly distinct from those related to TLR7 signaling, and points to specific inflammatory diseases that may benefit from inhibition of TLR8 in humans

Low surface expression of B7-1 (CD80) is an immunoescape mechanism of colon carcinoma

Artificially enforced expression of CD80 (B7-1) and CD86 (B7-2) on tumor cells renders them more immunogenic by triggering the CD28 receptor on T cells. The enigma is that such B7s interact with much higher affinity with CTLA-4 (CD152), an inhibitory receptor expressed by activated T cells. We show that unmutated CD80 is spontaneously expressed at low levels by mouse colon carcinoma cell lines and other transplantable tumor cell lines of various tissue origins. Silencing of CD80 by interfering RNA led to loss of tumorigenicity of CT26 colon carcinoma in immunocompetent mice, but not in immunodeficient Rag-/- mice. CT26 tumor cells bind CTLA-4Ig, but much more faintly with a similar CD28Ig chimeric protein, thus providing an explanation for the dominant inhibitory effects on tumor immunity displayed by CD80 at that expression level. Interestingly, CD80-negative tumor cell lines such as MC38 colon carcinoma and B16 melanoma express CD80 at dim levels during in vivo growth in syngeneic mice. Therefore, low CD80 surface expression seems to give an advantage to cancer cells against the immune system. Our findings are similar with the inhibitory role described for the dim CD80 expression on immature dendritic cells, providing an explanation for the low levels of CD80 expression described in various human malignancies

Autoimmune skin inflammation is dependent on plasmacytoid dendritic cell activation by nucleic acids via TLR7 and TLR9

Lupus-prone mice develop a chronic inflammatory response to cutaneous injury that depends on the production of type I interferon, TLR7, and TLR9

Blocking TLR7- and TLR9-mediated IFN-α Production by Plasmacytoid Dendritic Cells Does Not Diminish Immune Activation in Early SIV Infection

Persistent production of type I interferon (IFN) by activated plasmacytoid dendritic cells (pDC) is a leading model to explain chronic immune activation in human immunodeficiency virus (HIV) infection but direct evidence for this is lacking. We used a dual antagonist of Toll-like receptor (TLR) 7 and TLR9 to selectively inhibit responses of pDC but not other mononuclear phagocytes to viral RNA prior to and for 8 weeks following pathogenic simian immunodeficiency virus (SIV) infection of rhesus macaques. We show that pDC are major but not exclusive producers of IFN-α that rapidly become unresponsive to virus stimulation following SIV infection, whereas myeloid DC gain the capacity to produce IFN-α, albeit at low levels. pDC mediate a marked but transient IFN-α response in lymph nodes during the acute phase that is blocked by administration of TLR7 and TLR9 antagonist without impacting pDC recruitment. TLR7 and TLR9 blockade did not impact virus load or the acute IFN-α response in plasma and had minimal effect on expression of IFN-stimulated genes in both blood and lymph node. TLR7 and TLR9 blockade did not prevent activation of memory CD4+ and CD8+ T cells in blood or lymph node but led to significant increases in proliferation of both subsets in blood following SIV infection. Our findings reveal that virus-mediated activation of pDC through TLR7 and TLR9 contributes to substantial but transient IFN-α production following pathogenic SIV infection. However, the data indicate that pDC activation and IFN-α production are unlikely to be major factors in driving immune activation in early infection. Based on these findings therapeutic strategies aimed at blocking pDC function and IFN-α production may not reduce HIV-associated immunopathology. © 2013 Kader et al

Neutrophil extracellular traps mediate transfer of cytoplasmic neutrophil antigens to myeloid dendritic cells toward ANCA induction and associated autoimmunity.

Antineutrophil cytoplasmic antibodies (ANCAs) target proteins normally retained within neutrophils, indicating that cell death is involved in the autoimmunity process. Still, ANCA pathogenesis remains obscure. ANCAs activate neutrophils inducing their respiratory burst and a peculiar form of cell death, named NETosis, characterized by formation of neutrophil extracellular traps (NETs), decondensed chromatin threads decorated with cytoplasmic proteins endorsed with antimicrobial activity. NETs have been consistently detected in ANCA-associated small-vessel vasculitis, and this association prompted us to test whether the peculiar structure of NET favors neutrophil proteins uploading into myeloid dendritic cells and the induction of ANCAs and associated autoimmunity. Here we show that myeloid DCs uploaded with and activated by NET components induce ANCA and autoimmunity when injected into naive mice. DC uploading and autoimmunity induction are prevented by NET treatment with DNAse, indicating that NET structural integrity is needed to maintain the antigenicity of cytoplasmic proteins. We found NET intermingling with myeloid dendritic cells also positive for neutrophil myeloperoxidase in myeloperoxidase-ANCA-associated microscopic poliangiitis providing a potential correlative picture in human pathology. These data provide the first demonstration that NET structures are highly immunogenic such to trigger adaptive immune response relevant for autoimmunity