Summary Triggering receptor expressed on myeloid cells 1 (TREM‐1) is an activating receptor involved in inflammatory diseases and septic shock. The TREM‐1 ligand(s) (TREM‐1L) have not yet been identified. In this study, we performed a detailed analysis of the expression of mouse TREM‐1 and its ligand(s). Our results demonstrate that TREM‐1 is expressed on bone‐marrow‐derived dendritic cells (BMDC). On bone‐marrow‐derived macrophages (BMM) its expression is induced in vitro after stimulation by granulocyte–macrophage colony‐stimulating factor, interleukin‐3 or by myeloid differentiation primary response gene 88 (MyD88)‐dependent Toll‐like receptor (TLR) ligands. Under steady‐state conditions mouse TREM‐1 is detectable on a Gr‐1 − F4/80 + monocyte subpopulation bearing markers of resident monocytes, but not on Gr‐1 + F4/80 + inflammatory monocytes. During lipopolysaccharide (LPS)‐induced endotoxaemia TREM‐1 was also up‐regulated on inflammatory Gr‐1 + F4/80 + cells in vivo . In tumour‐bearing mice, TREM‐1 was up‐regulated on Gr‐1 + F4/80 + monocytes, which phenotypically and functionally resembled mononuclear myeloid‐derived suppressor cells. Using a soluble TREM‐1 fusion protein, we demonstrate that after intravenous injection of LPS TREM‐1L was induced on Gr‐1 + granulocytes and monocytes but not on other cell populations in peripheral blood. This up‐regulation on granulocytes was directly mediated by TLR ligands and required the adapter protein MyD88. In contrast to human, mouse platelets expressed TREM‐1L neither under steady‐state conditions nor after LPS injection in vivo . Our study reveals differential regulation of TREM‐1 expression on mouse monocyte subpopulations and improves our understanding of the biological role of TREM‐1 during disease.

Strategies of manipulating immunosuppressive regulatory T cells (Treg) in cancer patients are currently evaluated in clinical trials. Treg suppress immune responses of tumor‐specific T cells; yet, relatively little is known about the impact of Treg on innate immune cells in tumor models in vivo. Many tumors lose expression of MHC class I. Therefore, our study aimed at defining strategies to strengthen immune responses against a high tumor burden of the MHC class I‐deficient mouse lymphoma RMA‐S. We demonstrate that Treg depletion in mice led to tumor rejection that was dependent on T cells, NK cells and IFN‐γ. In the absence of Treg elevated levels of IFN‐γ were produced by tumor‐infiltrating T cells and NK cells. Tumor rejection observed in the absence of Treg correlated with a substantial IFN‐γ‐dependent increase in the numbers of tumor‐infiltrating leukocytes. The most abundant cell population in the tumors was macrophages. Tumor‐infiltrating macrophages from Treg‐depleted mice expressed increased amounts of MHC class II, produced highly enhanced levels of pro‐inflammatory cytokines and inhibited tumor cell proliferation. It was reported that tumor‐infiltrating macrophages have multi‐faceted functions promoting or counteracting tumor growth. In our study, high numbers of macrophages infiltrating RMA‐S tumors in the absence of Treg correlated with tumor rejection suggesting that macrophages are additional targets for Treg‐mediated immune suppression in cancer.

We showed previously that Tyk2(-/-) natural killer cells lack the ability to lyse leukemic cells. As a consequence, the animals are leukemia prone. Here, we show that the impaired tumor surveillance extends to T cells. Challenging Tyk2(-/-) mice with EL4 thymoma significantly decreased disease latency. The crucial role of Tyk2 for CTL function was further characterized using the ovalbumin-expressing EG7 cells. Tyk2(-/-) OT-1 mice developed EG7-induced tumors significantly faster compared with wild-type (wt) controls. In vivo assays confirmed the defect in CD8(+) cytotoxicity on Tyk2 deficiency and clearly linked it to type I IFN signaling. An impaired CTL activity was only observed in IFNAR1(-/-) animals but not on IFNgamma or IL12p35 deficiency. Accordingly, EG7-induced tumors grew faster in IFNAR1(-/-) and Tyk2(-/-) but not in IFNgamma(-/-) or IL12p35(-/-) mice. Adoptive transfer experiments defined a key role of Tyk2 in CTL-mediated tumor surveillance. In contrast to wt OT-1 cells, Tyk2(-/-) OT-1 T cells were incapable of controlling EG7-induced tumor growth.

Activation of immune cells has to be tightly controlled to prevent detrimental hyperactivation. In this regulatory process molecules of the C-type lectin-like family play a central role. Here we describe a new member of this family, CLEC12B. The extracellular domain of CLEC12B shows considerable homology to the activating natural killer cell receptor NKG2D, but unlike NKG2D, CLEC12B contains an immunoreceptor tyrosine-based inhibition motif in its intracellular domain. Despite the homology, CLEC12B does not appear to bind NKG2D ligands and therefore does not represent the inhibitory counterpart of NKG2D. However, CLEC12B has the ability to counteract NKG2D-mediated signaling, and we show that this function is dependent on the immunoreceptor tyrosine-based inhibition motif and the recruitment of the phosphatases SHP-1 and SHP-2. Using monoclonal anti-CLEC12B antibodies we found de novo expression of this receptor on in vitro generated human macrophages and on the human myelo-monocytic cell line U937 upon phorbol 12-myristate 13-acetate treatment, suggesting that this receptor plays a role in myeloid cell function.

IC31, the combination of a novel immunostimulatory oligodeoxynucleotide containing deoxy-Inosine/deoxy-Cytosine (ODN1a) and the antimicrobial peptide KLKL5KLK, represents a promising novel adjuvant signaling via the TLR9/MyD88-dependent pathway of the innate immune system. In mice, IC31 induces potent peptide-specific type 1 cellular immune responses, as well as mainly type 1 dominated protein-specific cellular and humoral immune responses. In addition, cytotoxic T lymphocytes were induced, able to kill efficiently target cells in vivo. Activation of murine dendritic cells by IC31 induced efficiently proliferation of naïve CD4+ TCR transgenic T cells (DO.11.10) as well as their differentiation into IFN-γ- and IL-4-producing T cells in vitro.

This study describes an entirely synthetic vaccine composed of antigenic peptides (T cell epitopes), oligodeoxynucleotides containing CpG-motifs (CpG-ODN) and poly-l-arginine (pR). CpG-ODN are known to be potent inducers of predominantly type 1-like immune responses, while polycationic amino acids, like pR, facilitate the uptake of antigens into antigen presenting cells (APCs). We demonstrate that the application of peptides and pR/CpG-ODN results in strongly enhanced peptide-specific immune responses as compared to the application of peptides with either of the immunomodulators alone. High numbers of antigen-specific T cells can be observed even after only one injection of the vaccine for a remarkably long period of time (at least 372 days). Furthermore, the potentially harmful systemic release of pro-inflammatory cytokines induced upon injection of CpG-ODN is inhibited. Thus, the combined application of CpG-ODN and pR may represent a novel vaccine strategy in humans.

Uso de una molecula de acido oligodesoxinucleico (ODN) que tiene la estructura segun la formula (I) en la que cualquier X es O u S. cualquier NMP es 2''-monofosfato o monotiofosfato de desoxinucleosido, seleccionado entre el grupo compuesto por monofosfato o monotiofosfato de desoxiadenosina-, desoxiguanosina-, desoxiinosina-, desoxicitosina-, desoxiuridina-, desoxitimidina-, 2-metil-desoxiinosina-, 5-metil-desoxicitosina-, desoxipseudouridina-, desoxirribosapurina-, 2-amino-desoxirribosapurina-, 6-S-desoxiguanina-, 2-dimetil-desoxiguanosina- o N-isopentenil-desoxiadenosina, NUC es un 2'' desoxinucleosido seleccionado entre el grupo compuesto por desoxiadenosina-, desoxiguanosina-, desoxiinosina-, desoxicitosina-, desoxiuridina-, desoxitimidina-, 2-metil-desoxiinosina-, 5-metil-desoxicitosina-, desoxipseudouridina-, desoxirribosapurina-, 2-amino-desoxirribosapurina-, 6-S-desoxiguanina-, 2-dimetil-desoxiguanosina- o N-isopentenil-desoxiadenosina, ayb son numeros enteros de 0 a 10 0 con la condicion de que la suma de a + b este entre 4 y 150, B y E son grupos comunes de los extremos 5'' o 3'' de moleculas de acido nucleico y se seleccionan independientemente entre el grupo compuesto por -H, -CH3, -COH, -COCH3, -OH, -CHO, -PO4, -PSO3, -PSO2, -PS3O, -PS4, -SO3, -PO4-(CH2)1-6-NH2 o -PO4-(CH2)1-6-NH-marcado, para preparar una composicion farmaceutica inmunoestimuladora.

ABSTRACT Four outer membrane proteins of Escherichia coli were examined for their capabilities and limitations in displaying heterologous peptide inserts on the bacterial cell surface. The T7 tag or multiple copies of the myc epitope were inserted into loops 4 and 5 of the ferrichrome and phage T5 receptor FhuA. Fluorescence-activated cell sorting analysis showed that peptides of up to 250 amino acids were efficiently displayed on the surface of E. coli as inserts within FhuA. Strains expressing FhuA fusion proteins behaved similarly to those expressing wild-type FhuA, as judged by phage infection and colicin sensitivity. The vitamin B 12 and phage BF23 receptor BtuB could display peptide inserts of at least 86 amino acids containing the T7 tag. In contrast, the receptors of the phages K3 and λ, OmpA and LamB, accepted only insertions in their respective loop 4 of up to 40 amino acids containing the T7 tag. The insertion of larger fragments resulted in inefficient transport and/or assembly of OmpA and LamB fusion proteins into the outer membrane. Cells displaying a foreign peptide fused to any one of these outer membrane proteins were almost completely recovered by magnetic cell sorting from a large pool of cells expressing the relevant wild-type platform protein only. Thus, this approach offers a fast and simple screening procedure for cells displaying heterologous polypeptides. The combination of FhuA, along with with BtuB and LamB, should provide a comprehensive tool for displaying complex peptide libraries of various insert sizes on the surface of E. coli for diverse applications.