Abstract
Type 1 diabetes (T1D) is an autoimmune disease wherein a loss in immune tolerance leads to the
initiation and progressive destruction of insulin-producing ß cells. Numerous immune interventions have been
reported to delay ß-cell loss, but a few clinical trials have demonstrated safety and efficacy. Arguably, Fc
receptor–nonbinding anti-CD3 monoclonal antibodies (anti-CD3 mAb) have been the most successful clinical
immunotherapy for T1D, showing promising clinical outcomes in patients with new onset T1D as well as high-
risk, nondiabetic relatives of T1D patients. However, a significant number of patients still did not respond
positively to anti-CD3 therapy and the duration of response is limited. Hence, there is a critical need to develop
novel therapeutic interventions to induce immune tolerance for the treatment of T1D. Programmed cell death
protein 1 (PD-1): PD-Ligand 1 (PD-L1) axis plays important roles in immune tolerance. NOD mice lacking PD-
L1 or PD-1 develop accelerated T1D and PD-1/PD-L1 expression is low in immune cells of both NOD mice and
T1D patients. Genetically engineering approaches to overexpress PD-L1 or PD-1 in immune cells have shown
to promote immune tolerance in T1D. However, there are still challenges in clinical applications of viral
engineered cells such as toxicity, genotoxicity, low engraftment of gene-edited cells. PD-1/PD-L1 axis also plays
a critical role in anti-CD3-mediated tolerance in T1D. Anti-CD3 therapy induces immune tolerance in overt T1D
in TGFß-dependent mechanisms, however CD3 antibody-induced remission of T1D in NOD mice is fully
reversed by PD-1/PD-L1 blockade, suggesting that the PD-1/PD-L1 axis may also play a critical role in the
response of T1D patients to anti-CD3 immunotherapy. Thus, combining anti-CD3 mAb with TGFb1/PD-L
treatment may more synergistically restore immune tolerance in T1D; however, this has not yet been tested.
Therefore, the overall goal of the proposed study is to develop a viral engineering- and cell-free strategy to deliver
TGFß1 and PD-L1 for inducing immune tolerance in T1D using extracellular vesicles (EVs) produced from
mesenchymal stem cells (MSCs). Our central hypothesis that MSC-derived EVs (MSC-EVs) engineered to
deliver TGFb1 and PD-L1 effectively induce immune tolerance in T1D and synergistically enhance anti-CD3-
mediated tolerance in T1D. Aim 1 is to define the mechanisms by which EVs carrying TGFb1 and PD-L1
suppress auto-reactive T cells. Aim 2 is to demonstrate the therapeutic effect of the engineered EVs in
prevention and reversal of T1D onset. Aim 3 is to examine the synergistic effect of the engineered EVs on anti-
CD3-mediated tolerance. The proposed project will provide EV-based therapy to induce immune tolerance and
help improve the efficacy and safety of anti-CD3 therapy in T1D. The significant preclinical data of the
engineered EVs in prevention and reversal of new-onset T1D will facilitate the development of robust and ready-
to-use MSC-EV therapeutics for T1D and other autoimmune diseases.