*1.4. Immune Checkpoint Molecules-Based Therapy*

Immune checkpoint molecules comprise a group of co-stimulatory and inhibitory proteins used to regulate the body's immune response within a specific microenvironment. Checkpoints such as CD28, a receptor commonly expressed on the surface of CD4<sup>+</sup> T cells, binds to one of two molecules (B7.1 and B7.2). This interaction promotes proliferation of CD4<sup>+</sup> T cells and subsequent migration towards designated target cells [105,106]. Similar processes exist with CD8<sup>+</sup> T cells, however such stimulatory action relies more heavily on the interaction between molecules such as CD70 and CD137 or CD134 [107]. Contrarily, T cell activation can be suppressed via inhibitory signaling pathways governed by checkpoint proteins including programmed cell death protein 1 (PD-1) and its cognate ligands, programmed cell death ligand 1 (PD-L1) and PD-L2 [108]. When bound to these ligands, PD-1 works to regulate the adaptive immune response by initiating immunosuppressive signals leading to the induction of apoptosis and reduced cell proliferation [109–111]. Further examples of co-inhibitory molecules include cytotoxic T lymphocytes-associated antigen 4 (CTLA-4), a CD28 homolog which binds competitively to B7.1/2. Similar to PD-1, CTLA-4 reduces T cell activation via inhibitory signaling pathways [112]. In either case, the relationships between these varied axes serve as mechanisms for acute and precise control of the body's immune system which poses potential solutions for autoimmune diseases like T1D.

In recent years, immune checkpoint therapy has revolutionized the field of oncology. Many cancer cells possess genetic and epigenetics irregularities allowing them to utilize immune checkpoints to promote survival. Studies have found that a variety of cancer cell types upregulate PD-L1 in response to interferon gamma (IFNγ) as well as other oncogenic signaling pathways [113,114]. Consequently, tumor cells express PD-L1 to abrogate T cell mediated antitumor responses. As a result, therapeutics have been developed to interrupt the PD-1/PD-L1 axis allowing T cells to function more effectively. Such therapeutics utilize monoclonal antibodies (mAb) targeted at immune checkpoints markers (PD-1, PD-L1, and CTLA-4) to inhibit T cell suppression [115,116]. However, immune checkpoint blockades have been linked to spontaneous development of autoimmune diseases. Examples of disease resulting from treatment with anti-PD-L1 antibodies include diabetes mellitus,

hepatitis, myasthenia gravis, sarcoidosis, hypothyroidism, endophthalmitis, and various skin rashes [117,118]. This relationship indicates that the absence of co-inhibitory signaling may increase the likelihood that an individual develops one of the aforementioned disorders. Several case studies have been conducted analyzing individuals who have developed late onset T1D in response to immune checkpoint therapy [118,119]. Recognizing this relationship, efforts have been made to assess whether increasing co-inhibitory signaling within pancreatic islets could increase beta cell survival.

Recent studies seeking to better understand T1D progression have determined that PD-L1 is expressed by insulin producing beta cells within pancreatic islets during insulitis. Upregulation of PD-L1 coincides with islet infiltration, as well as other factors such as increased exposure to interferons (IFN) alpha and gamma. Studies analyzing this relationship have determined that the heightened presence of IFNα and IFNγ activates STAT1 and STAT2 transcription factors. This activation corresponds with increased transcription of interferon regulatory factor 1 (IRF1) and subsequent PD-L1 upregulation by pancreatic beta cells [120,121]. Unfortunately, minimal research has been conducted to appraise the potential benefit of increasing PD-L1 or CTLA-4 expression in beta cells to enhance survival during T1D progression. However, preliminary research has demonstrated the protective effect of organ-specific PD-L1 expression in transgenic NOD mice. Wang et al. found that the severity of insulitis in PD-L1 transgenic NOD mice was significantly reduced when compared to controls [122]. Furthermore, islets transplanted into diabetic recipients persisted for a significantly longer period of time when compared to non-transgenic controls. Despite this, development of T1D remained constant between experimental and control groups. Another study attempting to increase survival rates among transplanted human islet-like organoids (HILOs) within NOD mice determined that overexpression of PD-L1 contributed significantly to the HILOs' survival rate within a diabetic mouse model. Without disturbing insulin production, PD-L1<sup>+</sup> HILOs maintained glucose homeostasis for more than 50 days whereas PD-L1- HILOs were only able to maintain glucose homeostasis for approximately 10 days [123]. These data present a potential therapeutic benefit to immune checkpoint therapy in T1D.

A significant roadblock when utilizing immune checkpoint proteins in a clinical setting stems from the mechanism by which PD-L1 and CTLA-4 overexpression is induced. One potential solution presents itself in the form of iron-oxide nanoparticles (NPs). Nanoparticles conjugated to various microRNAs (miRNAs) can be used to induce overexpression of co-inhibitory molecules for the purpose of protecting endogenous beta cells [124]. While the literature pertaining to this specific topic is limited, studies attempting to enhance cancer therapeutics have determined that PD-L1 regulation can be achieved via NPs conjugated to miR-200c. Such a combination has proven to inhibit PD-L1 expression, especially when compared to naked miR-200c [125]. Further examples of miRNAs which contribute to the regulation of PD-L1 and CTLA-4 include miR-138-5p, miR-513, miR-200a, and miR-34a [126–128]. NPs serve as an ideal delivery vehicle for miRNA-based therapeutic payloads [129]. Contrarily, NPs can be used to deliver antisense oligonucleotides designed to increase expression of co-inhibitory molecules such as PD-L1 and CTLA-4 within pancreatic islets. These immune checkpoint protein molecules currently under investigation have been tabulated together in Table 3 for a better overview.

Despite the potential benefits associated with immune checkpoint therapy in T1D, minimal research has been conducted to further its clinical application. This particular subset of biomolecular research remains dormant while alternative therapeutic avenues are explored. Utilizing the innate mechanisms by which the immune system is stimulated/inhibited could prove useful in the battle against T1D. In conjunction with more traditional forms of treatment, immune checkpoint therapy has the potential to curb the progression of T1D and preserve insulin independence for a more prolonged period of time. Furthermore, such applications may provide clinicians with a more effective form of theranostic-based treatment for T1D patients.


**Table 3.** Summary of strategies for identifying and targeting immune checkpoint markers towards T1D.
