Locally applied mesenchymal stromal cells (MSCs) have the capacity to promote the healing of perianal fistulas in Crohn's disease (CD) and are under clinical development for the treatment of... Show moreLocally applied mesenchymal stromal cells (MSCs) have the capacity to promote the healing of perianal fistulas in Crohn's disease (CD) and are under clinical development for the treatment of proctitis in ulcerative colitis (UC). Despite these clinical advances, the mechanism of action of local MSC therapy in inflammatory bowel disease (IBD) is largely unknown. We hypothesized that the local cytokine environment in IBD patients affects the immunomodulatory properties of MSCs. To evaluate this, 11 cytokines were analyzed in inflamed tissues obtained from CD and UC patients. Based on the identified cytokine profiles 4 distinct cytokine mixtures that mimic various inflammatory IBD environments were established. Next, MSCs were cultured in the presence of either of these 4 cytokine mixtures after which the expression of immunomodulatory and tissue regenerative molecules and the capacity of MSCs to modulate T-cell proliferation and dendritic cell (DC) differentiation were assessed. Our data show that MSCs respond, in a cytokine-specific manner, by upregulation of immunomodulatory and tissue regenerative molecules, including cyclooxygenase-2, indoleamine 2,3-dioxygenase, and transforming growth factor-beta 1. Functional studies indicate that MSCs exposed to a cytokine profile mimicking one of the 2 UC cytokine milieus were less effective in inhibition of DC differentiation. In conclusion, our data indicate that cytokine mixes mimicking the local cytokine milieus of inflamed UC colonic or CD fistulas tissues can differentially affect the immunomodulatory and tissue regenerative characteristics of MSCs. These data support the hypothesis that the local intestinal cytokine milieu serves as a critical factor in the efficacy of local MSC treatment. Show less
Schmitt, S.; Tahk, S.; Lohner, A.; Hanel, G.; Maiser, A.; Hauke, M.; ... ; Subklewe, M. 2020
Conventional dendritic cell (DC) vaccine strategies, in which DCs are loaded with antigens ex vivo, suffer biological issues such as impaired DC migration capacity and laborious GMP production... Show moreConventional dendritic cell (DC) vaccine strategies, in which DCs are loaded with antigens ex vivo, suffer biological issues such as impaired DC migration capacity and laborious GMP production procedures. In a promising alternative, antigens are targeted to DC-associated endocytic receptors in vivo with antibody-antigen conjugates co-administered with toll-like receptor (TLR) agonists as adjuvants. To combine the potential advantages of in vivo targeting of DCs with those of conjugated TLR agonists, we generated a multifunctional antibody construct integrating the DC-specific delivery of viral- or tumor-associated antigens and DC activation by TLR ligation in one molecule. We validated its functionality in vitro and determined if TLR ligation might improve the efficacy of such a molecule. In proof-of-principle studies, an alpha CD40 antibody containing a CMV pp65-derived peptide as an antigen domain (alpha CD40(CMV)) was genetically fused to the TLR5-binding D0/D1 domain of bacterial flagellin (alpha CD40.Flg(CMV)). The analysis of surface maturation markers on immature DCs revealed that fusion of flagellin to alpha CD40(CMV) highly increased DC maturation (3.4-fold elevation of CD80 expression compared to alpha CD40(CMV) alone) by specifically interacting with TLR5. Immature DCs loaded with alpha CD40.Flg(CMV) induced significantly higher CMVNLV-specific T cell activation and proliferation compared to alpha CD40(CMV) in co-culture experiments with allogeneic and autologous T cells (1.8-fold increase in % IFN-gamma/TNF-alpha(+) CD8(+) T cells and 3.9-fold increase in % CMVNLV-specific dextramer(+) CD8(+) T cells). More importantly, we confirmed the beneficial effects of flagellin-dependent DC stimulation using a tumor-specific neoantigen as the antigen domain. Specifically, the acute myeloid leukemia (AML)-specific mutated NPM1 (mNPM1)-derived neoantigen CLAVEEVSL was delivered to DCs in the form of alpha CD40(mNPM1) and alpha CD40.Flg(mNPM1) antibody constructs, making this study the first to investigate mNPM1 in a DC vaccination context. Again, alpha CD40.Flg(mNPM1)-loaded DCs more potently activated allogeneic mNPM1(CLA)-specific T cells compared to alpha CD40(mNPM1). These in vitro results confirmed the functionality of our multifunctional antibody construct and demonstrated that TLR5 ligation improved the efficacy of the molecule. Future mouse studies are required to examine the T cell-activating potential of alpha CD40.Flg(mNPM1) after targeting of dendritic cells in vivo using AML xenograft models. Show less
