TLR triggering on tolerogenic dendritic cells results in TLR2 up-regulation and a reduced proinflammatory immune program.

Tolerogenic dendritic cells (TDC) offer a promising therapeutic potential to ameliorate autoimmune diseases. Reported to inhibit adaptive immune responses, little is known about their innate immunity receptor repertoire. In this study, we compared three types of human TDC (IL-10-DC, dexamethasone (DX)-DC, and 1,25(OH)(2)D(3)-DC) by their TLR expression and response to a set of TLR ligands. TDC are endowed with the same TLR set as standard monocyte-derived dendritic cells but respond differentially to the TLR stimuli Pam3CSK4, polyinosinic-polycytidylic acid, LPS, and flagellin. TDC expressed low or no IL-12-related cytokines and remarkably elevated IL-10 levels. Interestingly, only TDC up-regulated the expression of TLR2 upon stimulation. This boosted the tolerogenic potential of these cells, because IL-10 production was up-regulated in TLR2-stimulated, LPS-primed DX-DC, whereas IL-12 and TNF-alpha secretion remained low. When comparing the TDC subsets, DX-DC and 1,25(OH)(2)D(3)-DC up-regulated TLR2 irrespective of the TLR triggered, whereas in IL-10-DC this effect was only mediated by LPS. Likewise, DX-DC and 1,25(OH)(2)D(3)-DC exhibited impaired ability to mature, reduced allostimulatory properties, and hampered capacity to induce Th1 differentiation. Therefore, both DX-DC and 1,25(OH)(2)D(3)-DC display the strongest tolerogenic and anti-inflammatory features and might be most suitable tools for the treatment of autoimmune diseases.

Vascular cell adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1) provide co-stimulation in positive selection along with survival of selected thymocytes.

T-cell differentiation in the thymus depends on positive selection of CD4+CD8+ double positive (DP) thymocytes by thymic major histocompatibility complex (MHC) molecules. Positive selection allows maturation of only those thymocytes that are capable of self-peptide-MHC recognition. Thymocytes that fail to bind self-peptide-MHC die by apoptosis. An important question in thymocyte differentiation is whether co-stimulation is required for positive selection and on which cells co-stimulatory molecules may be expressed in the thymus. The vascular cell adhesion molecule (VCAM-1) and the intercellular cell adhesion molecule (ICAM-1) are known to be potent co-stimulatory molecules in activation of peripheral T-cells by interacting with the integrins VLA-4 and LFA-1, respectively. We were prompted to investigate whether VCAM-1 and ICAM-1 may also act as co-stimulators during selection of thymocytes. By using recombinant proteins of murine VCAM-1 and ICAM-1 fused to the Fc region of human IgG1 (rVCAM-1, rICAM-1) we examined the capacity of VCAM-1 and ICAM-1 to act as co-stimulatory molecules in positive selection in vitro. Triggering the CD3/TCR complex together with co-stimulation applied by rVCAM-1 or rICAM-1 induced the generation of CD4+ single positive (SP) thymocytes from CD4+CD8+ DP thymocytes whereas either signal alone did not result in generation of CD4+ SP thymocytes. VCAM-1 and ICAM-1 act therefore as co-stimulatory molecules in thymocyte positive selection in vitro. The generation of CD4+ SP cells is accompanied by cell survival both when it was co-stimulated with rVCAM-1 and with rICAM-1. Importantly we show here that VCAM-1 expression in the murine thymus is restricted to cortical F4/80 positive hematopoietic antigen presenting cells (hAPC) present exclusively in the cortex whereas expression of ICAM-1 has been reported on the epithelium both in cortex and medulla. This suggests that not only the cortical epithelium may use the co-stimulatory molecule ICAM-1 to mediate positive selection, but also cortical hAPCs may contribute to positive selection of thymocytes by using the co-stimulator VCAM-1.

The glycosylation of thymic microenvironments. A microscopic study using plant lectins.

The thymus is the principal organ for development of T-cells. Thymocyte precursors from bone marrow-derived progenitor cells enter the thymus where they differentiate involving several differentiation stages into mature T-cells that can leave the thymus to the periphery. Migration of thymocytes through the thymus and their development are tightly controlled by the interaction of thymocytes with components of the thymic microenvironments. Several studies have demonstrated the pivotal importance of glycosylation in cell-cell interactions or interactions of cells with extracellular matrix components (ECM) in various physiologic processes in the body. The knowledge on glycosylation of thymic microenvironments is however limited although the presence of C-type lectin receptors such as DC-SIGN, mannose receptor and DEC-205, which are specifically recognizing distinct carbohydrate moieties emphasize the importance of glycosylation in the thymus. In order to outline the distribution of glycoconjugates in microenvironments of the human thymus we studied the glycosylation of the human thymic microarchitecture by using plant lectins in situ. Eleven plant lectin-biotin conjugates with distinct specificity were used and analyzed by fluorescence microscopy. Mannose glycoconjugates, specifically detected by the lectins GNA and NPA, were abundant in the cortex but not in the medulla. Dendritic cells present in the thymic cortex were specifically co-stained with the galactose-specific lectins DSA and PNA. Several lectins bound to the thymic vasculature. The alpha2-fucose-specific lectin UEA stained thymic blood vessels in the interlobular space and medulla and capillaries in the cortex. In addition to UEA, thymic blood vessels and capillaries also reacted with the lectins DSA, PNA and the alpha-GalNac-specific lectin HPA. In contrast, lymph vessels present in the interlobular space do not interact with UEA, DSA and PNA, but only with HPA, revealing a disparate glycosylation pattern of lymph and blood vessels that may be important to determine the direction of thymocytes entering or leaving the thymus. In conclusion, the restricted expression patterns of carbohydrates defined microenvironments in the human thymus highlight the importance of glycosylation in various steps of T-cell development.

Chimerism in kidneys, livers and hearts of normal women: implications for transplantation studies.

Tissue chimerism was recently described in transplanted organs from female donors into male recipients, by demonstration of the Y-chromosome in tissue-derived cells. It was claimed that these Y-chromosome positive cells were recipient derived. To find out whether the chimeric cells, derived from pregnancies of sons or blood transfusions, could have been present in the solid organs before transplantation, we performed the following study. In situ hybridization for the Y-chromosome was performed on the normal organs (51 kidneys, 51 livers, 69 hearts) from 75 women of the normal population, whose child and blood transfusion status were known. Chimeric cells were found in 13 kidneys, 10 livers and 4 hearts, of 23 women. There was no relation between the child status or the blood transfusion history with the presence of Y-chromosome positive cells. We have for the first time demonstrated that male cells are present in normal kidneys, livers and hearts. Theoretically, these organs could have been used for the transplantation. Therefore, our findings demonstrate that the chimeric cells thus far described in transplantation studies, are not necessarily donor derived, and could have been present in the organs before the transplantation.