T Cell Cancer Therapy Requires CD40-CD40L Activation of Tumor Necrosis Factor and Inducible Nitric-Oxide-Synthase-Producing Dendritic Cells

Effective cancer immunotherapy requires overcoming immunosuppressive tumor microenvironments. We\ua0found that local nitric oxide (NO) production by tumor-infiltrating myeloid cells is important for adoptively transferred CD8(+) cytotoxic T\ua0cells to destroy tumors. These myeloid cells are phenotypically similar to inducible nitric oxide synthase (NOS2)- and tumor necrosis factor (TNF)-producing dendritic cells (DC), or Tip-DCs. Depletion of immunosuppressive, colony stimulating factor 1 receptor (CSF-1R)-dependent arginase 1(+) myeloid cells enhanced NO-dependent tumor killing. Tumor elimination via NOS2 required the CD40-CD40L pathway. We also uncovered a strong correlation between survival of colorectal cancer patients and NOS2, CD40, and TNF expression in their tumors. Our results identify a network of pro-tumor factors that can be targeted to boost cancer immunotherapies

Immature mouse granulocytic myeloid cells are characterized by production of ficolin-B

Ficolins activate the lectin pathway of the complement system upon binding to carbohydrate patterns on pathogens. To characterize the producer cells of ficolin-B the expression of mouse ficolin-B, the orthologue of human M-ficolin, was studied in macrophages and dendritic cells during differentiation from bone marrow cells, in primary granulocytes, and during differentiation of granulocytes derived from ER-Hoxb8 cells. Expression of ficolin-B mRNA declined in all myeloid cell types to low levels during terminal differentiation. However, in contrast to macrophages and dendritic cells, ficolin-B expression was enhanced upon activation in granulocytes. High expression of ficolin-B was observed in primary immature neutrophilic CD11b+ Ly-6Cint Ly-6Ghigh granulocytes when isolated from the bone marrow, in particular during sepsis. Ficolin-B was demonstrated in lysates of primary granulocytes, ER-Hoxb8-derived granulocytes, bone marrow-derived macrophages, and dendritic cells. Native ficolin-B from cell lysates and supernatants of granulocytes activated the lectin pathway as measured by binding to MASP-2 and inducing C4 deposition. Specific staining demonstrated intra-cellular or cell associated ficolin-B protein in activated immature granulocytes deposited in a granular fashion. This study shows that ficolin-B is stored in and set free from immature granulocytic myeloid cells indicating a role in the early infection-induced cellular response of these inflammatory cells.Fil: Weber Steffens, Dorothea. Universitat Regensburg; AlemaniaFil: Hunold, Katja. Universitat Regensburg; AlemaniaFil: Kürschner, Johanna. Universitat Regensburg; AlemaniaFil: Giraldez Martinez, Sonia. Universitat Regensburg; AlemaniaFil: Elumalai, Preetham. Universitat Regensburg; AlemaniaFil: Schmidt, Dominic. Universitat Regensburg; AlemaniaFil: Trevani, Analía Silvina. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; ArgentinaFil: Runza, Valeria L.. Universitat Regensburg; AlemaniaFil: Männel, Daniela N.. Universitat Regensburg; Alemani

Targeting Tumor Cells with Anti-CD44 Antibody Triggers Macrophage-Mediated Immune Modulatory Effects in a Cancer Xenograft Model

<div><p>CD44, a transmembrane receptor reported to be involved in various cellular functions, is overexpressed in several cancer types and supposed to be involved in the initiation, progression and prognosis of these cancers. Since the sequence of events following the blockage of the CD44-HA interaction has not yet been studied in detail, we profiled xenograft tumors by RNA Sequencing to elucidate the mode of action of the anti-CD44 antibody RG7356. Analysis of tumor and host gene-expression profiles led us to the hypothesis that treatment with RG7356 antibody leads to an activation of the immune system. Using cytokine measurements we further show that this activation involves the secretion of chemo-attractants necessary for the recruitment of immune cells (i.e. macrophages) to the tumor site. We finally provide evidence for antibody-dependent cellular phagocytosis (ADCP) of the malignant cells by macrophages.</p></div

Tumor and host samples show distinct cytokine secretion profiles upon RG7356 treatment.

<p>(A) Cluster analysis of Luminex cytokine panel. Samples were analyzed at 8 and 168 hours after treatment in both human- and mouse-specific assays. Rows represent analytes, columns represent samples. Only analytes covered by both panels were used for clustering. Circles represent human samples, triangles represent mouse samples; open and solid symbols represent 8 and 168 hours of treatment, respectively, while color codes depict the different treatment groups. (B) and (C) Side-by-side comparison of human (left) and mouse (right) analyte concentrations (pg/ml) for the three different treatments. Shown are concentrations for GM-CSF (B) and MIP-1a (C).</p

The increased amounts of MIP-1a and MCP-1 decline to baseline levels after one week.

<p>Time-course study of MDA-MB231 xenografts over one week after treatment with RG7356. MIP-1a (A) and MCP-1 (B) concentrations were analyzed in obtained tumor lysates by ELISA.</p

RG7356 treatment leads to a deregulation of genes in both tumor and host at early time points.

<p>(A) Number of differentially expressed human and mouse transcripts at 3 different time points of anti-CD44 treatment bearing the IgG1 backbone compared to vehicle control. (B) Number of differentially expressed human and mouse transcripts at 3 different time points of anti-CD44 treatment bearing the IgG4 backbone compared to vehicle control. (C) Heatmap view of predicted activity of biological processes obtained from IPA® downstream processes analysis. Activated processes are shown in different shades of orange, inhibited processes are shown in different shades of blue. If no activity assessment was possible, the biological process is shown in grey.</p