Type 1 diabetes results from autoimmune destruction of the insulin-producing beta cells. Cell therapy with tolerogenic dendritic cells (tolDCs) may selectively modulate islet autoimmunity to interfere with beta-cell destruction. We previously showed that TolDCs induce antigen-specific Tregs that transfer tolerance by linked suppression in a tissue-specific manner. We have now upgraded the protocol to generate tolDC from monocytes by 1,25(OH)2 VitaminD3 and dexamethasone to clinical grade. The present study was designed to evaluate the stability of these tolDCs, examine their safety in vivo and obtain proof-of-concept evidence of their immunomodulatory properties.
First, we evaluated the durability of the phenotype and function of clinical-grade tolDCs in vitro. To examine whether tolDCs induce antigen-specific tolerance in vivo, HLA-DR4-Tg mice were treated with autologous tolDCs either unpulsed or pulsed with DR4-binding proinsulin peptide (C19-A3) and monitored for adverse events. One week before the readout, mice were challenged by immunization with intact human proinsulin and autoreactive IL-10 and IFNg responses measured.
The phenotype and function of tolDCs proved stable in vitro despite repeated stimulation with LPS, CD40L or inflammatory mediators, specified by expression of inhibitory molecule PD-L1 and low expression of HLA-DR and CD86. TolDCs retained high IL-10, low IL-12p70 production and the capacity to inhibit effector T cell proliferation. Vaccination of DR4-Tg mice with tolDCs proved safe. Importantly, IL-10 production to proinsulin increased after treatment with C19-A3-pulsed tolDCs. Moreover, subsequent immunization of DR4-Tg mice with proinsulin resulted in reduced IFNg production in mice treated with C19-A3-pulsed tolDCs compared to those treated with tolDCs only.
These data show that monocyte modulation with VitD3 and dexamethasone generates potent and stable tolerogenic DCs for clinical cell-therapy. Administration of C19-A3-pulsed tolDCs in humanized mice is safe and shows that tolDCs inhibit autoimmunity in vivo, paving the way for clinical evaluation of tolDC-vaccination in patients.