Anti-CCR7 (aa 2-22) polyclonal antibody

The chemokine receptor CCR7 plays a pivotal role in health and disease. In particular, CCR7 controls homing of antigen-bearing dendritic cells and T cells to lymph nodes, where adaptive immune responses are initiated. However, CCR7 also guides T cells to inflamed synovium and thereby contributes to rheumatoid arthritis and promotes cancer cell migration and metastasis formation. Nanobodies have recently emerged as versatile tools to study G-protein-coupled receptor functions and are being tested in diagnostics and therapeutics. In this study, we designed a strategy to engineer novel nanobodies recognizing human CCR7. We generated a nanobody library based on a solved crystal structure of the nanobody Nb80 recognizing the (2)-adrenergic receptor ((2)AR) and by specifically randomizing two segments within complementarity determining region 1 (CDR1) and CDR3 of Nb80 known to interact with (2)AR. We fused the nanobody library to one half of split-YFP in order to identify individual nanobody clones interacting with CCR7 fused to the other half of split-YFP using bimolecular fluorescence complementation. We present three novel nanobodies, termed Nb1, Nb5, and Nb38, that recognize human CCR7 without interfering with G-protein-coupling and downstream signaling. Moreover, we were able to follow CCR7 trafficking upon CCL19 triggering using Nb1, Nb5, and Nb38.

Tumor progression in the host leads to severe impairment of intrathymic T-cell differentiation/maturation, leading to the paralysis of cellular anti-tumor immunity. Such suppression manifests the erosion of CD4(+)CD8(+)double-positive (DP) immature thymocytes and a gradual increase in CD4(-)CD8(-)double negative (DN) early T-cell progenitors. The impact of such changes on the T-cell progenitor pool in the context of cancer remains poorly investigated. Here, we show that tumor progression blocks the transition of Lin(-)Thy1.2(+)CD25(+)CD44(+)c-Kit(low)DN2b to Lin(-)Thy1.2(+)CD25(+)CD44(-)c-Kit(-)DN3 in T-cell maturation, instead leading to DN2-T-cell differentiation into dendritic cells (DC). We observed that thymic IL-10 expression is upregulated, particularly at cortico-medullary junctions (CMJ), under conditions of progressive disease, resulting in the termination of IL-10R(high)DN2-T-cell maturation due to dysregulated expression of Notch1 and its target, CCR7 (thus restricting these cells to the CMJ). Intrathymic differentiation of T-cell precursors in IL-10(-/-)mice andin vitrofetal thymic organ cultures revealed that IL-10 promotes the interaction between thymic stromal cells and Notch1(low)DN2-T cells, thus facilitating these DN2-T cells to differentiate toward CD45(+)CD11c(+)MHC-II(+)thymic DCs as a consequence of activating the Ikaros/IRF8 signaling axis. We conclude that a novel function of thymically-expressed IL-10 in the tumor-bearing host diverts T-cell differentiation toward a DC pathway, thus limiting the protective adaptive immune repertoire.