Biomaterials-based nanoparticles conjugated to regulatory T cells provide a modular system for localized delivery of pharmacotherapeutic agents.

Type 1 diabetes (T1D) presents with two therapeutic challenges: the need to correct underlying autoimmunity and restore ß-cell mass. We harnessed the unique capacity of regulatory T cells (Tregs) and the T cell receptor (TCR) to direct tolerance induction along with tissue-localized delivery of therapeutic agents to restore endogenous ß-cell function. Specifically, we designed a combinatorial therapy involving biomaterials-based poly(lactic-co-glycolic acid) nanoparticles co-loaded with the Treg growth factor, IL-2, and the ß-cell regenerative agent, harmine (a tyrosine-regulated kinase 1A [DYRK1A] inhibitor), conjugated to the surface of Tregs. We observed continuous elution of IL-2 and harmine from nanoparticles for at least 7 days in vitro. When conjugated to primary human Tregs, IL-2 nanoparticles provided sufficient IL-2 receptor signaling to support STAT5 phosphorylation for sustained phenotypic stability and viability in culture. Inclusion of poly-L-lysine (PLL) during nanoparticle-cell coupling dramatically increased conjugation efficiency, providing sufficient IL-2 to support in vitro proliferation of IL-2-dependent CTLL-2 cells and primary murine Tregs. In 12-week-old female non-obese diabetic mice, adoptive transfer of IL-2/harmine nanoparticle-conjugated NOD.BDC2.5 Tregs, which express an islet antigen-specific TCR, significantly prevented diabetes demonstrating preserved in vivo viability. These data provide the preclinical basis to develop a biomaterials-optimized cellular therapy to restore immune tolerance and promote ß-cell proliferation in T1D through receptor-targeted drug delivery within pancreatic islets.

Clinical Applications of Regulatory T cells in Adoptive Cell Therapies.

Interest in adoptive T-cell therapies has been ignited by the recent clinical success of genetically-modified T cells in the cancer immunotherapy space. In addition to immune targeting for malignancies, this approach is now being explored for the establishment of immune tolerance with regulatory T cells (Tregs). Herein, we will summarize the basic science and clinical results emanating from trials directed at inducing durable immune regulation through administration of Tregs. We will discuss some of the current challenges facing the field in terms of maximizing cell purity, stability and expansion capacity, while also achieving feasibility and GMP production. Indeed, recent advances in methodologies for Treg isolation, expansion, and optimal source materials represent important strides toward these considerations. Finally, we will review the emerging genetic and biomaterial-based approaches on the horizon for directing Treg specificity to augment tissue-targeting and regenerative medicine.

Expansion of Human Tregs from Cryopreserved Umbilical Cord Blood for GMP-Compliant Autologous Adoptive Cell Transfer Therapy.

Umbilical cord blood is a traditional and convenient source of cells for hematopoietic stem cell transplantation. Thymic regulatory T cells (Tregs) are also present in cord blood, and there is growing interest in the use of autologous Tregs to provide a low-risk, fully human leukocyte antigen (HLA)-matched cell product for treating autoimmune diseases, such as type 1 diabetes. Here, we describe a good manufacturing practice (GMP)-compatible Treg expansion protocol using fluorescence-activated cell sorting, resulting in a mean 2,092-fold expansion of Tregs over a 16-day culture for a median yield of 1.26 × 109 Tregs from single-donor cryopreserved units. The resulting Tregs passed prior clinical trial release criteria for Treg purity and sterility, including additional rigorous assessments of FOXP3 and Helios expression and epigenetic analysis of the FOXP3 Treg-specific demethylated region (TSDR). Compared with expanded adult peripheral blood Tregs, expanded cord blood Tregs remained more naive, as assessed by continued expression of CD45RA, produced reduced IFN-γ following activation, and effectively inhibited responder T cell proliferation. Immunosequencing of the T cell receptor revealed a remarkably diverse receptor repertoire within cord blood Tregs that was maintained following in vitro expansion. These data support the feasibility of generating GMP-compliant Tregs from cord blood for adoptive cell transfer therapies and highlight potential advantages in terms of safety, phenotypic stability, autoantigen specificity, and tissue distribution.