Dendritic cells (DC) are critically involved in initiating adaptive immune responses but they also play a pivotal role in promoting and maintaining tolerance. We identified DC-10, a subset of human tolerogenic DC characterized by the ability to produce high levels of IL-10. DC-10 can be generated in vitro from monocytes in the presence of IL-10 and are currently used for generating CD4+ T regulatory type 1 (Tr1) cells for Treg cell-based therapies. The aims of my PhD project are: 1)To define the molecular signature of DC-10; 2)To investigate modulation of in vitro and in vivo allogeneic T cell responses by DC-10. To achieve the first aim, we compared DNA microarray-based transcriptional profiling of in vitro generated DC-10 and of immunogenic mature (mDC) or immature DC (iDC). DC-10 display a molecular signature of immuno-modulation and anti-inflammation with up-regulated IL-10- and Tissue Growth Factor (TGF) beta-dependent pathways, and down-modulated signaling of pro-inflammatory cytokines. Moreover, DC-10 have an interesting matrix remodeling profile that places them as hypothetical determinants of the tolerogenic environment in peripheral tissues. Through the screening of gene differentially expressed, we also identified two DC-10-specific markers, CD141 and CD163 that in combination with CD14 and CD16 allow their identification and isolation in vivo. CD141+CD163+CD14+CD16+ cells isolated from peripheral blood of healthy donors produce IL-10 at steady state and upon activation, in the absence of IL-12, and prime allogeneic naïve CD4+ T cells. Furthermore, preliminary results indicate that allogeneic naïve CD4+ T cells primed with ex vivo isolated DC-10 produce IL-10 when re-stimulated with the same allo-antigen. To achieve the second aim, we studied the ability of DC-10 to modulate allogeneic CD8+ T cells. DC-10 activate allogeneic CD8+ T cells at lower levels compared to mDC, as demonstrated by the expression of activation markers and IFN-γ production. Despite the expression of HLA class I and co-stimulatory molecules, DC-10 poorly stimulate the proliferation of allogeneic CD8+ T cells, which is partially rescued by the addition of exogenous IL-12. Moreover, DC-10 inhibit mDC-induced proliferation of allogeneic CD8+ T cells in a dose-dependent manner, suggesting an active mechanism of suppression mediated by DC-10. This immunomodulatory effect is contact-independent, as shown by transwell experiments. In parallel, we assessed the ability of DC-10 to prime allogeneic T cells in vivo in NSG mice co-injected with DC-10 and allogeneic PBMC. Upon in vitro re-stimulation of spleen cells with allo-antigens, CD4+ and CD8+ T cells from mice injected with DC-10 proliferate less compared to T cells isolated from control mice injected with mDC. Conversely, response to 3rd party allo-antigens is comparable. These data indicate that DC-10 prime T cells in vivo and induce anergic allo-antigen specific T cells. In conclusion, we identify the biomarkers of DC-10 that allow their study in vivo in healthy and pathological conditions. Moreover, we demonstrate the ability of DC-10 to modulate allogeneic CD8+ T cell responses, supporting the use of DC-10 as DC-based therapy in transplantation. DC-10 may indeed promote tolerance via allo-specific CD4+ Tr1 cells, and, concomitantly, limit allo-reactive CD8+ T cell responses.

(2016). Unravelling the fingerprint and regulatory functions of human tolerogenic DC-10. (Tesi di dottorato, Università degli Studi di Milano-Bicocca, 2016).

Unravelling the fingerprint and regulatory functions of human tolerogenic DC-10

COMI, MICHELA
2016

Abstract

Dendritic cells (DC) are critically involved in initiating adaptive immune responses but they also play a pivotal role in promoting and maintaining tolerance. We identified DC-10, a subset of human tolerogenic DC characterized by the ability to produce high levels of IL-10. DC-10 can be generated in vitro from monocytes in the presence of IL-10 and are currently used for generating CD4+ T regulatory type 1 (Tr1) cells for Treg cell-based therapies. The aims of my PhD project are: 1)To define the molecular signature of DC-10; 2)To investigate modulation of in vitro and in vivo allogeneic T cell responses by DC-10. To achieve the first aim, we compared DNA microarray-based transcriptional profiling of in vitro generated DC-10 and of immunogenic mature (mDC) or immature DC (iDC). DC-10 display a molecular signature of immuno-modulation and anti-inflammation with up-regulated IL-10- and Tissue Growth Factor (TGF) beta-dependent pathways, and down-modulated signaling of pro-inflammatory cytokines. Moreover, DC-10 have an interesting matrix remodeling profile that places them as hypothetical determinants of the tolerogenic environment in peripheral tissues. Through the screening of gene differentially expressed, we also identified two DC-10-specific markers, CD141 and CD163 that in combination with CD14 and CD16 allow their identification and isolation in vivo. CD141+CD163+CD14+CD16+ cells isolated from peripheral blood of healthy donors produce IL-10 at steady state and upon activation, in the absence of IL-12, and prime allogeneic naïve CD4+ T cells. Furthermore, preliminary results indicate that allogeneic naïve CD4+ T cells primed with ex vivo isolated DC-10 produce IL-10 when re-stimulated with the same allo-antigen. To achieve the second aim, we studied the ability of DC-10 to modulate allogeneic CD8+ T cells. DC-10 activate allogeneic CD8+ T cells at lower levels compared to mDC, as demonstrated by the expression of activation markers and IFN-γ production. Despite the expression of HLA class I and co-stimulatory molecules, DC-10 poorly stimulate the proliferation of allogeneic CD8+ T cells, which is partially rescued by the addition of exogenous IL-12. Moreover, DC-10 inhibit mDC-induced proliferation of allogeneic CD8+ T cells in a dose-dependent manner, suggesting an active mechanism of suppression mediated by DC-10. This immunomodulatory effect is contact-independent, as shown by transwell experiments. In parallel, we assessed the ability of DC-10 to prime allogeneic T cells in vivo in NSG mice co-injected with DC-10 and allogeneic PBMC. Upon in vitro re-stimulation of spleen cells with allo-antigens, CD4+ and CD8+ T cells from mice injected with DC-10 proliferate less compared to T cells isolated from control mice injected with mDC. Conversely, response to 3rd party allo-antigens is comparable. These data indicate that DC-10 prime T cells in vivo and induce anergic allo-antigen specific T cells. In conclusion, we identify the biomarkers of DC-10 that allow their study in vivo in healthy and pathological conditions. Moreover, we demonstrate the ability of DC-10 to modulate allogeneic CD8+ T cell responses, supporting the use of DC-10 as DC-based therapy in transplantation. DC-10 may indeed promote tolerance via allo-specific CD4+ Tr1 cells, and, concomitantly, limit allo-reactive CD8+ T cell responses.
GRANUCCI, FRANCESCA
GREGORI, SILVIA
Dendritic; Cells,; Immunological; Tolerance,; IL-10
BIO/12 - BIOCHIMICA CLINICA E BIOLOGIA MOLECOLARE CLINICA
Italian
Scuola di Dottorato in Medicina Traslazionale e Molecolare
SCUOLA DI DOTTORATO IN MEDICINA TRASLAZIONALE E MOLECOLARE (DIMET) - 72R
28
2014/2015
(2016). Unravelling the fingerprint and regulatory functions of human tolerogenic DC-10. (Tesi di dottorato, Università degli Studi di Milano-Bicocca, 2016).
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/10281/137397
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