Disease-modifying pharmacological treatments of type 1 diabetes: Molecular mechanisms, target checkpoints, and possible combinatorial treatments

1. TxNIP assembly and transcription

Hyperglycemia plays a major role in β-cell loss, but the pathways leading to destructive effects of glucotoxicity remain unclear. Thioredoxin-interacting protein (TxNIP) was recently established as a novel proapoptotic gene in the β-cells that is significantly overexpressed during hyperglycemia in T1D (Minn et al, 2005), and its expression in β-cells is directly elevated due to the toxic effects of continuous exposure to elevated glucose (Fig. 2). TxNIP binds to the antioxidant enzyme thioredoxin (Txn) and inhibits it, consequently facilitating oxidative damage in β-cells and is recognized as one of the key therapeutic targets of cell stress (Choi and Park, 2023) responsible for activating NLRP3 inflammasome (Qayyum et al, 2021). In pancreatic β-cells, glucose-stimulated TxNIP transcription is mediated by a glucose-sensitive transcription factor, carbohydrate response-element binding protein (ChREBP; Shalev, 2014). Two major isoforms of ChREBP, α and β, can have opposing metabolic effects in regulating β-cell function. Although CHREBPα can have protective effects during oxidative stress, CHREBPβ, in particular, is the predominant isoform highly elevated in conditions of hyperglycemia and diabetes, leading to β-cell death (Katz et al, 2022). One way to prevent TxNIP elevation in the β-cell is to target the nuclear-cytoplasmic shuttling of ChREBP and binding to DNA, which is regulated by PKA- and AMPK-mediated phosphorylations. AMPK is the central regulator of cell energy homeostasis, and upon activation, it phosphorylates several downstream effectors responsible for glucose metabolism and cell longevity. Metformin in particular was shown to transiently inhibit complex I of the electron transport chain in the mitochondrion, leading to activation of LKB1-mediated AMP-activated protein kinase (AMPK; Mihaylova and Shaw, 2011). Metformin-activated AMPK directly hinders hyperglycemia-stimulated nuclear translocation of ChREBP and forkhead box O1 (FOXO1), preventing their subsequent recruitment on TxNIP promoter (Li et al, 2015).

2. Endoplasmic reticulum stress and unfolded protein response

Among other hyperglycemia-induced impairment pathways (Marré and Piganelli, 2017; Thomaidou et al, 2020), key contributors of β-cell dysfunction in T1D are endoplasmic reticulum (ER) stress and the aberrant unfolded protein response (UPR; Roep et al, 2021; Sahin et al, 2021). The ER contains the largest calcium reserve in the cell and is responsible for maintaining a variety of translation chaperones and enzymes that facilitate protein folding, maturation, and other posttranslational modifications described in detail elsewhere (Anken and Braakman, 2005). Under stimuli caused by transient stressors, β-cells activate UPR’s function and promote cell survival. However, exposure to prolonged or chronic stress, commonly associated with diabetic autoimmune and metabolic dysfunction, UPR adaptive response is not sufficient and leads to direct β-cell apoptosis and premature loss of β-cell mass. In line with this concept, the UPR is commonly associated with the regulation of β-cell function, generation of antigenic peptides, and development of T1D (Fig. 2) (Thomaidou et al, 2018).

3. Glutamate-induced β-cell damage

During diabetes, chronic exposure to hyperglycemia leads to glucotoxicity through rapid matrix acidification of the β-cells and control of the membrane potential of β-cells by affecting the ratio of intracellular ATP/ADP concentrations, elevation of intracellular glutamate, and acceleration of β-cell damage via NMDAR (Huang et al, 2017). Extended glutamate stimulation of NMDARs elevates intracellular mitochondrial accumulation of calcium, thereby laying the groundwork for ER dysfunction and oxidative stress (Fig. 2) (Mohsin et al, 2020), eventually culminating in β-cell apoptosis (Feissner et al, 2009). Considering the electrical activity of the β-cells in disease led Sterk and colleagues to discover that inhibition of NMDARs in human islets can improve the survival of endocrine cells and restore GSIS (Šterk et al, 2021). Marquard and colleagues presented a further series of genetic and pharmacological experiments and found that inhibiting NMDARs, particularly with dextromethorphan (DXM), can significantly improve GSIS through prolonging depolarized state and plateau phase of calcium oscillations (Marquard et al, 2015). These findings were later supported in non-obese diabetic (NOD) mice as a model for T1D, highlighting DXM as a novel safe intervention candidate to be used as adjunct treatment of preclinical (stage 2) or recent-onset T1D (stage 3; Wörmeyer et al, 2024).

1. Incretin rescue of GSIS

Binding of incretins such as glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide-1 (GLP-1) initiates cyclic adenosine monophosphate (cAMP) downstream signaling, which is recognized as the most significant potentiator of insulin secretion (Fig. 3). Canonical GLP-1R signaling initiates the activation of adenylate cyclase (AC) and rapid production of cAMP, which potentiates GSIS by 2 distinct protein-kinase A (PKA)-dependent and -independent mechanisms (Seino et al, 2009). Activated PKA downstream signaling is responsible for many processes related to calcium regulation, such as phosphorylating Kir6.2 and SUR1 K_ATP channel subunits (Béguin et al, 1999), stimulation of L-type voltage-gated calcium channels (Bünemann et al, 1999), and calcium release from the intracellular calcium stores such as the ER. PKA-independent mechanism potentiating insulin secretion occurs by cAMP-initiated activation of cAMP-binding protein Epac2 (Epac2), which was recently discovered to regulate the insulin granule density by engaging Ras-like small GTPase Rap1 (Fig. 1) (Shibasaki et al, 2007). These pathways are especially important, as a rescue mechanism for the endocrine system during hyperglycemic conditions, as GLP-1R signaling initiates several metabolic mechanisms that can not only have a short-term effect on β-cell function, such as potentiation of GSIS, but also regulate long-term effects, such as β-cell survival and proliferation. Potentiating GSIS via GLP-1R-induced signaling can play a crucial role in rescuing the endocrine system short term during hyperglycemic conditions. However, incretin-induced signaling can also have long-term effects, such as regulating the survival and growth of β-cells.

2. Autophagic pathway for cell survival

Autophagy is a catabolic cellular process for recycling damaged molecules and subcellular elements via lysosome-mediated degradation. It is fundamental for appropriate β-cell function and survival during stressful conditions, including ER stress and pathogenic activity of lysosomal cathepsin proteases, leading to the assembly of highly antigenic epitopes for CD4+ T cells (Reed et al, 2021; Crawford et al, 2022). Many groups hypothesized that defective autophagy and lysosome defects are associated T1D pathogenesis and can directly affect β-cell presentation and expression of MHC class I and class II molecules (Russell et al, 2019) in β-cells (Fierabracci, 2014; Muralidharan et al, 2021; Mohammadi-Motlagh et al, 2023). As a therapeutic target, impaired autophagy can be the driving force behind general β-cell metabolic dysfunction in T1D (Muralidharan et al, 2021), especially considering the importance of lysosomes’ role in the cellular response to stress (Lawrence and Zoncu, 2019). Pharmaceutical regulation of the mTORC1 pathway signaling was found to regulate autophagy during metabolic stress in β-cells and can present a specific addressable target in T1D (Bartolomé et al, 2014; Blandino-Rosano et al, 2017; Pasquier et al, 2019). As such, GLP-1R-cAMP-induced signaling has a significant effect in regulating β-cell autophagy (Kanasaki et al, 2019). There are numerous reports on how GLP-1R agonist (GLP-1RA) can restore lysosomal function, relieve the impairment in autophagic flux, and promote β-cell proliferation and autophagy specifically under metabolic stress (Fig. 2) (Wang et al, 2015a; Zummo et al, 2017; Arden, 2018). Limited data are available regarding DPP-4 inhibitor (DPP-4i) effect on stimulating autophagy in pancreatic cells (Zheng et al, 2018).

3. Wnt/β-catenin pathway-induced β-cell proliferation and survival

Wnt-β-catenin pathway in the β-cell is responsible for a broad spectrum of processes, including β-cell vitality and proliferation, GSIS, and function and secretion of incretin hormones (Napolitano et al, 2023). In pancreatic β-cells, Wnt-β-catenin pathway is activated by Wnt frizzled receptors and phosphatidylinositol 3-kinase (PI3K)-AKT-mTORC1 downstream signaling. Both cascades lead to the inhibition of glycogen synthase kinase 3β (GSK3β) and the accumulation of free cytosolic β-catenin (Ju et al, 2006; Liu and Habener, 2008). Free β-catenin moves from cytosol into the nucleus and combines with T-cell factor (TCF) family of transcription factors, such as TCF7L2, thereby activating the expression of Wnt signaling target β-cell proliferative genes, including cyclin D1 and c-myc (Fig. 3). A noncanonical cAMP-PKA-mediated phosphorylation of β-catenin Ser-675 independently of GSK3β was reported by a number of research groups in different cell types by activated protein kinases (eg, P21-activated protein kinase), insulin, GLP-1, and IGF-1 (Hino et al, 2005; Chiang et al, 2013; Chiang and Jin, 2014).

In particular, Liu and colleagues elucidated a mechanism in pancreatic cells by which GLP-1RA-mediated activation of cAMP-PKA phosphorylates β-catenin on Ser-675 and thereby enhances the expression of TCF7L2 (Liu and Habener, 2008). More recently, GLP-1RA-induced noncanonical β-catenin pathway of Ser-675 phosphorylation was observed in combination with GABA. Shao and colleagues (Shao et al, 2020) specifically investigated GLP-1 and GABA pathways after discovering that a DPP-4 inhibitor sitagliptin in combination with GABA increased β-cell mass in the STZ-induced T1D mouse model (Liu et al, 2017). It was found that β-cell oxidative damage induced by high glucose and characterized by elevated expression of TxNIP can be attenuated by GABA, dependent on GLP-1R stimulation. A question was raised by the group on the role of the GABA and specifically GABA_B receptor in β-catenin-mediated signaling with additional stimulation of GLP-1R. An elegant study by Rachdi and colleagues in part addresses this question, as they find that the expression of functional GABA_B receptors in human islets is directly induced by GLP-1-cAMP signaling, which consequently initiates GABBR2 subunit mRNA expression (Rachdi et al, 2020). In clinical applications of combination therapies with DPP-4i, when the goal is to increase GLP-1R-mediated signaling, insufficient endogenous GLP-1 elevation could be an issue affecting treatment efficacy (Griffin et al, 2014).

4. Gastrin-induced increase in β-cell mass and islet cell transdifferentiation

Increasing functional β-cell mass is one of the primary goals for intervention therapies in T1D. Proton-pump inhibitors (PPIs) are substituted benzimidazoles that inhibit acid secretion by blocking the hydrogen (H+) potassium (K+) adenosine triphosphate enzyme system (“proton pump”) in a variety of cells (Shin and Sachs, 2008), leading to a reduction in stomach acid production and an elevation in the blood levels of gastrin. Gastrin has pronounced effect on β-cell mass regeneration and reprogramming of pancreatic ductal cell pool and plays a substantial role in the process of differentiation of Neurogenin+ cells into endocrine cells during neonatal development (Gu et al, 2002; Collombat et al, 2006; Suissa et al, 2013). Possible mode of action of gastrin in the endocrine cells can be related to its function as both a conventional gut hormone and a powerful neurotransmitter in the gastrointestinal tract, central nervous system (CNS), and peripheral neurons. In human CNS and pancreas, gastrin primarily targets widely expressed G_i-coupled CCKB receptor (Lee et al, 1993; Saillan-Barreau et al, 1999; Reubi et al, 2003) inducing antiapoptotic PKC-independent activation of mitogen-activated protein kinases (MAPK) and PI3K-AKT-mTOR signaling cascades (Kowalski-Chauvel et al, 1996; Ramamoorthy et al, 2004). During embryonic development, gastrin and CCKB receptors are highly expressed throughout the fundamental stage of endocrine cell differentiation in the developing pancreas, but their expression decreases after birth along with β-cell neogenesis (Bardram et al, 1990; Suissa et al, 2013; Téllez and Montanya, 2014). Gastrin expression is also peaked during the differentiation of human stem cells into insulin-secreting cells (Suissa et al, 2013), and gastrin-producing syngeneic mesenchymal stem cells was found to preserve β-cell mass in NOD mice (Gaudreau et al, 2022). These observations correspond with earlier studies that gastrin-induced signaling may induce β-cell regeneration in the NOD mouse model when coadministered with EGF (Suarez-Pinzon et al, 2005; Wang et al, 2012) and GLP-1RA (Fig. 3) (Suarez-Pinzon et al, 2008; Sasaki et al, 2015). Gastrin-related signaling pathways and biological activities in various cellular processes are reviewed in more detail elsewhere (Rehfeld, 2019; Zeng et al, 2020).

1. Nonspecific immune therapy with ATG

The polyclonal antithymocyte globulins (ATG) in the form of purified IgG fraction of sera from horses or rabbits immunized with thymocytes or T-cell lines have been used as potent and broad immune therapy for several decades (Mohty, 2007), primarily not only in organ transplantation but also in some autoimmune indications (Fig. 4). ATGs have a nonspecific and extensive antibody specificities ranging from direct immune response antigens to adhesion and cell trafficking ligands and finally heterogeneous pathways targets that can be found elsewhere (Gaber et al, 2010). Besides a well documented T-cell depletion caused by ATG directly targeting CD3, its nonexhaustive target list also includes HLA class I molecules expressed on all nucleated cells, CD2, CD4, CD5, CD6, CD8, CD25, and CD28 predominant in T-cells, CD80, CD86, and CTLA-4 in APCs, including several specific targets on activated B lymphocytes, such as CD5, CD20, CD28, and CD30. HLA class II molecule is, of course, a particularly interesting T1D target for ATG to explore in more detail, as its expression by β-cells as well as on APCs is hypothesized to initiate islet autoimmunity in disease pathophysiology. With such a broad spectrum of nonspecific immunosuppression, it is important to note that at low, submitogenic concentrations, therapy with ATG facilitates antibody-dependent cell cytotoxicity of preactivated T cells, but not resting cells.

Mechanistically, ATG-induced T-cell depletion is driven by antibody-dependent cytotoxicity and apoptotic Fas/Fas-L signaling pathway in mature lymphocytes (Genestier et al, 1998; Lagunas-Rangel, 2023). Earlier studies revealed that ATG activation-induced cell death (AICD) in mature-activated T cells could be initiated by continuous simultaneous signaling of monoclonal anti-CD3 antibodies and mitogenic lectins such as phytohemagglutinin (PHA; Kabelitz et al, 1993). Importantly, Fas-induced apoptosis required ATG preparation to include CD2 and CD3 antibodies for simultaneous stimulation, and the ATG-driven T-cell depletion was prevented by cyclosporin A, FK506, and rapamycin. Hypothetically, in a clinical setting, an adjustment in ATG dosing and composition can potentially be tailored to selectively target activated T cells and avoid substantial adverse events such as lymphocyte depletion and subsequent immunodeficiency. As such, low dosing of ATG and ATG in combination with pegylated granulocyte colony-stimulating factor (ATG + GCSF) in recent-onset T1D patients significantly reduced HbA1c. Additionally, ATG, but not ATG + GCSF, slowed the depletion of C-peptide at 1-year posttreatment (Haller et al, 2018). After 2 years of therapy, the levels of C-peptide were greater than placebo only in the ATG group, and both ATG and ATG + GCSF groups had significantly decreased levels of HbA1c (Haller et al, 2019). As a potential future treatment for T1D, it is important to note that low-dose ATG is not without significant limitations due to adverse events related to cytokine release or serum sickness. Although the side-effect profile of low-dose ATG is known and can generally be regulated, the intensity of serum sickness worsens with repeated use and may necessitate the use of follow-up treatments to maintain the outcome of the initial intervention. Development of innovative humanized ATG products holds promise to decrease occurrences of adverse events and improve clinical application as an intervention strategy.

2. Specific immune therapy targeting CD3 with teplizumab

Teplizumab, an FcR-nonbinding anti-CD3 monoclonal antibody (anti-CD3), has been shown to induce alterations in the differentiation and activation state of CD3-expressing T-cells in the circulating pool, instead of depleting them. Teplizumab is one of the most widely investigated immune therapies in the field of T1D, with an overall of 14 ongoing, terminated, and finished studies accessible at https://clinicaltrials.gov/. In 2022, it was the first approved drug for the delay of T1D stage 3 onset in adults and children aged ≥8 years. A recent analysis by Herold and colleagues (Herold et al, 2023) showed significant preservation of C-peptide in response to teplizumab therapy based on pooled data from 5 clinical trials, a total of 609 patients (375 treatment/234 control) with stage 3 T1D. The same study conducted a safety analysis of 6 clinical trials with a total of 1018 patients (treatment 787/control 233) with stage 2 or 3 T1D. Almost all patients experienced grade 1 or 2 adverse events with the vast majority resolving without additional interventions; however, severe adverse events (grade 3) took place in 59% of treated patients compared with 22.9% of the control group. It is important to note that, as was the case with other immune therapies, although C-peptide depletion was significantly reduced after teplizumab intervention, the treatment was not able to increase or fully preserve it.

Notably, the complete mechanism of action of teplizumab has not yet been fully elucidated. Several research groups examined changes in T-cell subsets and phenotypes of 18 favorably responding stage 3 T1D from Antibody for Tolerance in Recently Diagnosed Type 1 Diabetes (AbATE) study with a total of 77 patients (52 teplizumab and 25 control) after 2 courses of teplizumab therapy, 1 year apart (Long et al, 2016, 2017; Tooley et al, 2016). Poststudy analyses discovered elevated CD8+ central memory T cells with particular gene signatures characterized by high levels of PD-1, TIGIT, KLRG1, and other eomesodermin (EOMES)-associated transcription factors correlated with successful response to teplizumab intervention. In the following sections, we will carefully consider that these anergic CD4+ and exhausted CD8+ T cells share phenotype similarities aligning with known mechanisms of tolerance and exhaustion in cancers (Schietinger and Greenberg, 2014).

More specifically, it was discovered that complex effects of anti-CD3 lead to transient rises in PD-1+Foxp3+ Treg cells, exhausted CD57-KLRG1+PD-1+, TIGIT+KLRG1+, EOMES CD8 T cells, and anergic CD57-KLRG1-PD-1+ CD4+ T cells. Rapid upregulation of programmed death protein (PD-1), also referred to as CD279, restricts both adaptive and innate immune responses, such as proliferation and survival in activated T cells (Keir et al, 2007) by blocking CD3/CD28-mediated PI3K/AKT (Parry et al, 2005), ZAP70/CD3ζ (Sheppard et al, 2004), and Ras/MEK/Erk signaling pathways (Patsoukis et al, 2012). Importantly, PD-1/PD-1L axis is crucially involved in tumor progression because cancer cells can exploit it to evade the immune system (Han et al, 2020). Blocking inhibitory signals that are important in cancer immunotherapy, but PD-1/PD-1 ligand pathway inhibition can also lead to T1D (Hughes et al, 2015; Roep et al, 2021). Given the important role of PD-1 overexpression to tumor-specific T-cell immunity, it might be plausible to follow-up on patients exhibiting an increase in PD-1 in response to anti-CD3 treatment, paying specific attention to documented cancer-associated PD-1 polymorphisms: PD-1.1 (rs36084323), PD-1.3 (rs11568821), PD-1.5 (rs2227981), PD-1.9 (rs2227982), and PD-1 (rs7421861; Salmaninejad et al, 2018).

Teplizumab-induced elevated expression of inhibitory immunoreceptor TIGIT is primarily associated with limiting antitumor responses in lymphocytes. It is expressed on APCs and competes with CD226 in NK and T cells, similar to how CTLA-4 prevents CD80 and CD86 receptors from interacting with CD28 and promotes direct T-cell inhibitory signaling cascades (Manieri et al, 2017). TIGIT expression and signaling in T cells have been shown to inhibit T-cell priming and reduce TCR-driven activation signals (Joller et al, 2011). Limited available research shows teplizumab-induced TIGIT expression in CD8+ T cells only. Lack of TIGIT in Tregs post-teplizumab treatment is an important immune target to consider for future intervention strategies because earlier research linked TIGIT expression in Tregs with direct inhibition of Th1 and Th17 responses (Johnston et al, 2014; Joller et al, 2014), reduction of interferon gamma (IFN-γ) in patients with multiple sclerosis, potentially by suppressing IL-12-induced PI3K-AKT-mTOR signaling cascade responsible for inducing dysfunctional proinflammatory Th1 Treg program (Ouyang et al, 2012; Lucca et al, 2019).

Finally, teplizumab-induced killer cell lectin-like receptor subfamily G member 1 (KLRG1) expression was found to be elevated in both CD8+ and CD4+ T cells. Consistent with CD57-KLRG1+PD-1+, TIGIT+KLRG1+ CD8 T cells described as displaying a partially exhausted phenotype (Long et al, 2016), Li and colleagues established that KLRG1+ T cells exhibit abundant proinflammatory cytokines and high level of Foxp3 expression but were found to contribute to the impaired antitumor immunity of memory T cells (Li et al, 2016). Additionally, in accordance with previous observations on the viral role in the manifestation of T1D and observations from the AbATE study on an increase in the detectable viral load in the treated group, Long and colleagues found that Epstein-Barr virus (EBV) reactivation significantly correlated with elevated transcription of EOMES genes in CD8+ T cells (Long et al, 2016). These findings demonstrate that pharmacologically induced alterations of phenotypes that lead to expansion of partially exhausted T cells, although known to be detrimental in tumor immunology and viral containment, might be considered advantageous reactions to immune therapy in T1D. An ongoing study on long-term safety for teplizumab (NCT04598893) is currently underway as an extension of the PRV-031-001 (PROTECT) phase 3 study to evaluate the efficacy and safety in stage 3 children and adolescents aged 8 through 17.

1. Abatacept-induced costimulatory blockade of CD28-CD80/86 signaling axis

Abatacept is a cytotoxic T-lymphocyte-associated protein Ig (CTLA4Ig) that prevents costimulatory signaling in T cells via CD80 and CD86 axis and substantially limits CD4+ and CD8+ T-cell activation. Mechanistically, abatacept inhibits T-cell activation via binding to CD80 and CD86 on APCs, thereby blocking the costimulatory interaction with CD28 on T cells. Another target is an inducible T-cell co-stimulator (ICOS) that becomes rapidly expressed on activated CD4+ and CD8+ T cells together with CD28 and CTLA-4, indicating its role in regulating the adaptive T-cell response (Watanabe et al, 2005). A 2-year clinical study on costimulation modulation in 112 patients (abatacept: 77 patients, placebo: 35 patients) with stage 3 recent-onset T1D showed a significant delay in the reduction of C-peptide area under the curve (AUC) (aggregate P = .0022) and HbA1c (aggregate P = .002; Orban et al, 2011). Insulin dosage decrease was significant at 6 and 12 months but leveled out to match the placebo at 24 months. In this trial, grade 3 and higher adverse events were recorded for 18 patients treated with abatacept, with the majority experiencing mild grade 1 or 2 adverse events, with an overall rate of all adverse events similar to that of placebo. A follow-up of these patients 1 year later showed that costimulatory blockade with abatacept decelerated the deterioration of β-cell activity and most notably sustained reduction in HbA1c levels (P < .005) 3 years after the diagnosis of T1D (Orban et al, 2014). In terms of prevention, an abatacept clinical study was conducted on 81 stage 1 T1D patients (abatacept: 35 patients, placebo: 46 patients), where the disease progression was not significantly delayed, but abatacept reduced PD-1+ T-follicular helper (Tfh) cells (P < .0001), increased naive CD4+ T cells, and also reduced the frequency of Tregs from the baseline (P = .0067), with complete return to baseline in 1 year after treatment. It is worth noting that the abatacept prevention trial had a primary endpoint effect on glucose tolerance instead of C-peptide AUC (Russell et al, 2023).

Clinical findings in abatacept prevention trial showed that abatacept primarily reduced the frequency of activated ICOS+ T follicular (Tfh) and T peripheral (Tph) helper CD4+ T cells, while maintaining the total number of CD4+ and CD8+ T cells in the naive state. Tfh CD4+ T cells, in particular, are crucial for B-cell responses, including the formation of memory B cells and the generation of antibodies and autoantibodies. Curiously, the investigators did not see a reduction in insulin autoantibodies and other autoantibodies after abatacept treatment. However, the decrease in ICOS+ CD4+ T cells indicated that costimulation blockage might have inhibited T-cell activation overall. Importantly, although abatacept effectively reduced the frequency of activated CD4+ T cells by blocking the CD28 axis, it did not induce either full or partial exhaustion of T cells. In both pediatric and adult patients, the frequency of these cells returned to baseline levels 12 months after treatment.

2. Alefacept-induced costimulatory blockade of costimulatory CD2 signaling axis

Alefacept is a fusion protein composed of 2 LFA-3 molecules bound to IgG1 that binds CD2, expressed on CD4+ and CD8+ effector memory T cells, thereby interrupting CD2-mediated costimulatory activation. Inducing Remission in New-Onset T1D with alefacept (T1DAL; Rigby et al, 2013) study in stage 3 T1D patients (alefacept: 33 patients, placebo: 16 patients) showed a significant increase in the mean 4-hour C-peptide AUC (P = .019) and reduction in insulin demand (P = .02) after 12 months of treatment with no difference in HbA1c and mean 2-hour C-peptide AUC. All patients experienced at least 1 adverse event, mostly grade 1 or 2, some related to infection and/or major hypoglycemic events, 14 patients in alefacept group suffered from grade 3 and/or 4 adverse events compared with 9 taking placebo. Twenty-four months after 2 12-week treatments with alefacept, Rigby and colleagues subsequently reported on significant preservation of C-peptide AUC and reduction of exogenous insulin use in patients from the T1DAL study (Rigby et al, 2015).

Importantly, total white blood counts remained unchanged in both groups, but total lymphocytes and CD4+ and CD8+ T cells showed modest declines during the first and second course of treatment in the alefacept group, which rebounded by 78 weeks. The treatment significantly depleted CD4+ and CD8+ central memory and effector memory T cells, which were partially recovered at 24 months of follow-up. Importantly, alefacept therapy did not change the frequency of Tregs during and after the treatment. The 24-month findings of the T1DAL study support that a short-term focused immune intervention during the early stages may sustain pancreatic function in stage 3 recently diagnosed patients. Importantly, due to targeting particular T-cell memory subsets, alefacept treatment did not lead to cytokine release syndrome after administration and the reactivation of EBV, which was the case with some patients treated with anti-CD3 monoclonal antibodies (Keymeulen et al, 2010; Ambery et al, 2014).

1. GLP-1R-mediated short-term rescue of GSIS by elevating G_s-GPCR-cAMP-PKA/Epac2- and PDX1-associated cell function

GSIS is one of the fundamental regulatory mechanisms of insulin exocytosis in the β-cell that presents a key pharmaceutical target in diabetes. As discussed earlier, incretins initiate G_s-GPCR-cAMP signaling by phosphorylation by cAMP-dependent protein kinase A (PKA), and the cAMP-binding protein Epac2 (Seino et al, 2009). Two widely available antidiabetic therapeutic agents, GLP-1R agonists and DPP-4 inhibitors, have been developed with a particular focus on cAMP-induced signaling in β-cells. Mechanistic targets of these drugs can be found on Fig. 3. Studies on GLP-1-cAMP-PKA-induced signaling show that GLP-1RA can activate and upregulate the expression of pancreatic duodenal homeobox-1 protein (PDX1) responsible for β-cell phenotype and function (Wang et al, 2001). When used individually, the GLP-1RA efficacy in T1D adults has been assessed in 11 clinical trials with moderate beneficial effects on reduction in HbA1c levels and insulin demands (Tan et al, 2023). Importantly, GLP-1RA did not increase the incidence of serious adverse events, such as severe hypoglycemia or diabetic ketoacidosis. Together, these findings coupled with an understanding of pathways to be targeted by GLP-1RA make these drugs appealing candidates for a combination treatment of T1D.

2. Targeting G_i/o-GPCR signaling to amplify PI3K-AKT survival pathways

Today, there are several pharmacologically addressable G_i/o-GPCRs that are expressed on β-cells, such as somatostatin, gastrin/cholecystokinin (CCKB) receptors, α2A-adrenergic receptors (ADRs), D2-like dopamine receptors (DRDs), and GABA_B receptors. G_i/o-GPCR-initiated signaling inhibits cAMP transmission but is responsible for a significantly higher generation of PI3K, a lipid kinase that spreads intracellular signal cascades and regulates a variety of cellular processes. PI3K recruits protein kinase B (AKT), a downstream effector of multiple growth, proliferation, survival, metabolism, and autophagy pathways, such as mTORC1 (Jean and Kiger, 2014). A recent study by Dickerson and colleagues illuminates a primary mechanism of how G_i/o-GPCRs control β-cell electrical excitability by inducing membrane hyperpolarization, thereby directly affecting intracellular calcium handling and insulin secretion (Dickerson et al, 2022). The mechanism describes the regulation of electrogenic NKAs by GPCR-induced signaling, whereby G_s-GPCR-cAMP-PKA activation effectively inhibits NKA function, leading to membrane depolarization and calcium influx. Stimulation of G_i/o-GPCRs and tyrosine kinases (STKs, insulin receptors) on the contrary, hyperpolarizes cell membrane via activation of NKAs and initiates closure of voltage-gated calcium channels (depicted in Figs. 1 and 3). Understanding these processes is particularly important because it will allow a tailored intervention targeted specifically at 2 counter-regulatory mechanisms that regulate β-cell function, electrical excitability, and calcium handling by modulating NKA activity.

3. Competitively inhibiting glucagon receptor signaling

On another end of the pharmaceutical intervention spectrum are GPCR antagonists. Glucagon receptor is G_s-GPCR and acts through cAMP-PKA signaling as a part of paracrine crosstalk in the islets. Earlier works identified glucagon receptor antagonism as a promising pathway for overcoming complete insulin deficiency and maintaining appropriate glycemic control in animal models (Gelling et al, 2003; Wang et al, 2015b). Human immunoglobulin G2 (IgG2) mAb volagidemab competitively blocks glucagon receptors and inhibits its downstream signaling (Pettus et al, 2022). volagidemab 12-week treatment in 18–65 years T1D patients (volagidemab 35 mg: 2 patients, volagidemab 70 mg: 2 patients, placebo: 27 patients) failed to reduce the daily insulin use but showed a significant reduction in HbA1c and tolerable safety profile. Importantly, all participants had a fasting C-peptide of <0.7 ng/mL, and although authors note no increase in hypoglycemia in treated patients, significant increases in serum transaminases, LDL cholesterol, and blood pressure were recorded (Pettus et al, 2022).

4. Inhibiting G_s-GPCR to amplify AMPK survival pathway

Blunting glucagon downstream signaling by means of reducing cAMP accumulation and activation of AMP-activated protein kinase (AMPK) is a known mechanism of another antidiabetic drug: metformin (Miller et al, 2013; Cao et al, 2014). The detailed glucoregulatory mechanisms and modes of action of metformin are extensively discussed elsewhere (Foretz et al, 2019, 2023). Under cellular energy deficit, AMPK deactivates biosynthetic pathways and other nonessential processes that consume ATP and activates alternative catabolic pathways that generate ATP to restore energy balance (Hardie, 2014). Suppression of mitochondrial complex 1 results in a reduction in ATP levels and an elevation in the AMP/ATP ratio, which subsequently directly inhibits gluconeogenesis (Fig. 3) (Foretz et al, 2010).

Previous studies have demonstrated that metformin exerts its effects through mechanisms that are both AMPK-dependent and AMPK-independent by impeding mitochondrial respiration, typically activated by G_s-GPCR-cAMP downstream signaling (Owen et al, 2000), and inhibiting lysosome-related mechanisms (Rena et al, 2017). Independently of AMPK signaling, metformin acts as a potent mTORC1 inhibitor (Kalender et al, 2010), conceivably by means of inhibition of V-ATPase-Ragulator complex located in lysosomes and endosomes (Ma et al, 2022). It is an intriguing therapeutic target to consider for managing cellular metabolism and energy stress in the islets as is required for LKB1-mediated AMPK and mTORC1 activation, thereby providing a switch between catabolism and anabolism in the cell (Zhang et al, 2014). Although mechanistic targets of metformin in pancreatic islets still have not been completely elucidated, some studies have shown their protective and proliferative functions in β-cells in vitro (Marchetti et al, 2004; Tajima et al, 2017).

1. Calcium-induced inhibition of TxNIP assembly in β-cells

Xu and colleagues show that 1 way to significantly decrease proapoptotic TxNIP expression in β-cells under glucotoxicity can be achieved by using first-generation calcium channel blockers, such as verapamil and diltiazem (Xu et al, 2012). The authors indicate that downregulation of TxNIP expression is primarily mediated by overall downregulation of cytoplasmic calcium levels, nonspecific to class or type of a particular calcium channel blocker used (Fig. 4). Verapamil, specifically, was found to prevent nuclear transcription of TxNIP in the β-cell by blocking off E-box motif that serves as a binding site for ChREBP, significantly reducing its nuclear presence. Importantly, verapamil was shown to selectively reduce TxNIP expression only under hyperglycemic conditions, making it a promising intervention in patients suffering from diabetes.

Importantly, these findings were later successfully translated into clinical studies with verapamil showing improvement in β-cell function of recent-onset pediatric (Forlenza et al, 2023) and adult patients (Ovalle et al, 2018). In a more recent study, Xu and colleagues also discovered that verapamil-induced downregulation of TxNIP promotes the elevation of insulin-growth factor 1 (IGF-1) and a significant decrease in the expression of IGF-binding protein 3 (IGFBP3; Xu et al, 2023). The authors note the particular significance of the verapamil-enhanced IGF-1 signaling pathway in restoring appropriate β-cell function. This is an important observation, because although insulin receptor (IR) and IGF-1 receptor are highly homologous and share PI3K-AKT and RAS-MAPK signaling pathways, each receptor is responsible for distinct activities within the cell (Cai et al, 2017). IR is responsible for cell metabolism, whereas IGF-1-induced signaling controls mTORC1 phosphorylation of 4EBP1 protein responsible for growth and proliferation and p70 ribosomal protein S6 kinase (S6K1), mitigating oxidative stress, ribosomal, and mitochondrial biogenesis (Proud, 2007). IGFBP3 was recently found to be elevated in diagnosed recent-onset and established T1D patients and its direct binding of TMEM219 receptor initiates caspase-8 induced β-cell dysfunction and apoptosis (D’Addio et al, 2022). Therefore, a calcium blockade by pharmaceutical intervention can effectively target not 1, but several β-cell fragilities associated with hyperglycemia-induced metabolic stress through the downregulation of proapoptotic factor TxNIP and reduce extracellular proapoptotic signaling via IGFBP3. Considering these particular signaling mechanisms can also be beneficial when identifying combinational treatment synergies for other pharmaceutical agents that are also able to downregulate TxNIP expression and elevation of IGF-1, such as GLP-1RA (Xie et al, 2014).

2. Calcium blockade in immune cells

Calcium transport holds a central role in immunomodulation of T-cell function and adaptive immunity as a whole. The beneficial effects of L-type calcium channel blocker verapamil in T1D clinical trials and its effect on endocrine cells are described earlier in the paper. The outstanding question to consider is whether it might also have a role to play in immunomodulation through targeting Ca2+ transmission in immune cells. Importantly, the primary source of Ca2+ in T-cells is the Ca2+ release-activated Ca2+ channel (CRAC), which includes ORAI1 and ORAI2 proteins (Prakriya et al, 2006; Vaeth et al, 2017b). Although ORAI channels are resistant to verapamil, secondary Ca2+ sources, including T and L-type VGCC, have also been proposed to contribute to calcium-driven signaling cascades in T-cells (Fenninger and Jefferies, 2019). Altogether, these calcium fluxes are essential for the recruitment and accumulation of the nuclear factor of activated T-cells (NFAT) isoforms, as well as other transcription factors regulating clonal growth, cell survival, differentiation, and cytokine synthesis (Feske, 2007; Vaeth et al, 2017a).

Veytia-Bucheli and colleagues provide a comprehensive in vitro study on its role in T-cells with several interesting discoveries that can significantly contribute to increasing efficacy in future clinical studies (Veytia-Bucheli et al, 2022). In particular, the authors found that dose-dependent administration of verapamil in response to CD3 stimulation alone exerts complete inhibition of T-cell proliferation, and potently inhibits the expression of CD25, CD40L, and CD69 surface ligands. It was also found to impair the polarization of Th1, Th17, and Th2 along with the secretion of associated cytokines (eg, interferon gamma, TNF-α, IL-2, IL-17, IL-4, and IL-10). Most notably, this inhibitory effect is significantly negated when the cells are stimulated with both CD3 and CD28. These findings demonstrate that costimulatory signaling in T cells activates supplementary pathways that circumvent TCR-activated calcium-dependent processes that are restricted by verapamil, thereby partially recovering T-cell activity. Building on the existing promising clinical findings of verapamil intervention in recent-onset T1D patients (Ovalle et al, 2018; Forlenza et al, 2023), these in vitro discoveries provide opportunities for more focused pharmaceutical intervention strategies, such as including costimulatory signal blockers (eg, abatacept) in combination therapies that include verapamil as one of the components.

1. Mediating cytokine-induced damage in β-cells

Some groups have identified a detailed activation pathway of the ER stress sensors in the β-cells, particularly in response to inflammatory cytokines (Gysemans et al, 2008; Moore et al, 2011). IFN-γ triggers downstream signaling, typically via Janus kinase (JAK) that directly induces apoptosis via the activation of the signal transducer and activator of transcription (STAT) and binding to IFN-γ regulatory factor-1 (IRF-1) promoter. Importantly, IFN-γ upregulates the expression of MHC class I expression on the surface of β-cells (Baldeón et al, 1997); however, direct blockade of IFN-γ receptors on the β-cells did not impact diabetes progression (Thomas et al, 1998). This may be indicative of other interferons and γ-chain cytokines signaling through the JAK-STAT axis in β-cells and its importance in the pathogenesis of autoimmune diabetes. Mechanistically, inhibitors of JAK1/JAK2 inhibited JAK-STAT pathway in mouse and human β-cells and successfully blocked the effect of cytokines by downregulating MHC class I and reversing diabetes in NOD mice (Trivedi et al, 2017; Ge et al, 2020). Notably, these results successfully translated into a phase 2 clinical study, as daily treatment with a single tablet of baricitinib for over 48 weeks successfully preserved C-peptide in recent-onset T1D patients and reduced exogenous basal and prandial insulin intake with minimal adverse events (Waibel et al, 2023). The subsequent sections of this review article covering JAK-STAT-related autoimmune interventions in T1D provide a more comprehensive analysis of findings in Waibel and colleagues’ study. At present, 4 JAK inhibitors, namely baricitinib, tofacitinib, upadacitinib, and ruxolitinib, received approval for clinical use in the treatment of other indications, namely RA and myelofibrosis.

2. Blocking interferon gamma and γ chain cytokines signaling in immune cells

JAK inhibitors exert large potential as a comprehensive intervention for T1D, as they are uniquely positioned to address immune as well as endocrine dysfunctional JAK-STAT molecular pathways involved in disease pathophysiology. This is reflected in a recently published baricitinib phase 2 clinical study on 91 recent-onset T1D patients aged between 10 and 30 years old (treatment: 60 patients, placebo: 31 patients; Waibel et al, 2023). Most notably, at the 48-week assessment, the mixed-meal-stimulated C-peptide in baricitinib group was significantly higher than placebo (P = .001) and, importantly, higher than at baseline. Secondary endpoints also showed lower levels of HbA1c compared with placebo and nonsignificant reduction in insulin demand compared with baseline and placebo. Authors hypothesize that the treatment efficacy may be higher in patients with more residual C-peptide function, meaning initiating treatment immediately after diagnosis or preventatively in asymptomatic stage 2 patients. Interesting observations were made on mechanistic effects of baricitinib in immune cells. Waibel and colleagues recorded a significant reduction in the number of CD8+ T-cells and IL-21–stimulated phosphorylated STAT3. These observations are consistent with previous studies of JAK inhibitors in animal models (Trivedi et al, 2017; Ge et al, 2020). JAK inhibitors were seen to block JAK-STAT downstream signaling cascades initiated by T1D-associated IFN-γ and γ chain cytokines, including IL-21 (Sutherland et al, 2013; Ge et al, 2020).

Importantly, baricitinib is an orally administered tablet with an acceptable safety profile, which is additionally confirmed by a consensus statement for the treatment of immune-mediated inflammatory diseases with JAK inhibitors (Nash et al, 2021). The authors attribute low drug toxicity to partial inhibition of JAK at the highest doses of baricitinib and no effect on JAK at lower levels. These data combined suggest that JAK inhibitors, including baricitinib, have broad target ranges through diverse JAK-STAT roles regulating inflammation in immune and β-cells. As a result, these inhibitors appear to be more successful in combating the development of T1D compared with selective pharmaceutical ligands or cytokine blockers.

1. Preventing degradation of incretins and associated G_S-GPCR signaling in β-cells

As mentioned earlier, DPP-4i are widely used antidiabetic agents primarily administered to T2D patients to prevent the degradation of incretins, such as GIP and GLP-1, by DPP-4 enzymes. The importance of incretin-stimulated G_s-GPRC signaling in β-cells is described earlier in this review (see section Incretin rescue of GSIS). A meta-analysis on DPP-4i used as intervention therapy in 253 diagnosed T1D patients pooled from 5 randomized controlled trials showed a trend to decrease in HbA1c levels and improvement in insulin demands and glycemic control (Wang et al, 2018). Despite inconclusive clinical benefits when used as an individual component, DPP-4i are associated with a low incidence of minor adverse events and a high safety profile in adults, elderly, and pediatric patients (Gadsby, 2009; Jalaludin et al, 2022; Shankar et al, 2022; Nagao et al, 2023). In T2D clinical trials, sitagliptin specifically has been consistently observed to lower HbA1c and retain a high safety profile not only as a monotherapy, but in combination with metformin, pioglitazone, sulfonylurea, and insulin (Charbonnel et al, 2006; Raz et al, 2006; Rosenstock et al, 2006; Vilsbøll et al, 2010; Moses et al, 2016).

2. Pharmaceutically inhibiting the costimulatory CD26/mDPP-4 signaling axis in CD4+ T cells

CD26 is a T-cell costimulatory activation antigen with membrane-bound dipeptidyl peptidase-4 (mDPP-4) involved in various cellular processes through binding to several ligands, including adenosine deaminase (ADA), CD45, fibronectin, plasminogen, and caveolin-1 (Huang et al, 2022). CD26 is not only limited to T-cell functions but is also involved in B-cell activation (Bühling et al, 1995). Increasing evidence indicates that DPP-4 activity in the serum and mDPP-4 expression in immune cells play a role in the pathophysiology of autoimmune diseases (Yoon et al, 2021). As with other costimulatory pathways, such as CD28, costimulation of mDPP-4 in combination with an antigen-specific stimulus provided by a TCR-CD3 complex leads to maximal T-cell activation (Tanaka et al, 1994; ElTanbouly and Noelle, 2021).

Although data on DPP-4i impact in immune cells of T1D patients remain limited, Varga and colleagues discovered that in T1D patients, serum DPP-4 enzymatic activity is elevated, but mDPP-4 expression on lymphocyte membranes is reduced, which may indicate a novel T effector cell regulatory malfunction (Varga et al, 2011). Notably, serum DPP-4 levels in T1D patients were also found to correlate with diabetes duration (Wang and Hiesinger, 2013; Hirao et al, 2020). A number of recent studies show the involvement of CD26-mediated signaling in the differentiation of CD4+ T-cells into Th1 Th2 and Th17 subtypes, thereby promoting the secretion of IL-2, interferon gamma, IL-6, IL-17, and IL-22 inflammatory cytokines (Pinheiro et al, 2017; Zhao et al, 2021). Meta-analysis of 8 controlled T2D clinical trials showed that DPP-4i (sitagliptin and vildagliptin) are associated with significant reductions in TNF-α, an important mediator for T1D-related inflammatory processes (Atkin et al, 2017). Consistent with these findings and earlier studies on DPP-4i-induced alteration of the T-cell phenotype in T2D patients (Aso et al, 2015), Wang and colleagues show that inhibition of CD26 with sitagliptin in patients with LADA (20 sitagliptin, 20 control) significantly upregulates the ratio of Tregs to CD4+ T cells (P = .016) and downregulates the percentage of Th2 at 6 months (P = .015), 12 months (P = .012) and Th17 at 12 months (P = .020) of treatment (Wang et al, 2019).

3. Immune cell processes triggered by mDPP-4 activation

Regarding T1D-driven autoimmunity, mDPP-4 expression is highest in activated Th17 and Th1 and to a lesser extent in Th2 CD4+ T-cell subsets, presenting particular targets for pharmaceutical intervention (Willheim et al, 1997; Lun et al, 2007; Bengsch et al, 2012). In CD4+ T cells, ligation of mDPP-4 and CD45 complex provides a costimulatory signal for T-cell activation that enhances the downstream phosphorylation of CD3-ζ, p56lck (LCK), and ZAP-70 (Ishii et al, 2001; Katz et al, 2017).

Additionally, mDPP-4 forms complexes with ADA, thereby significantly depleting the local concentration of adenosine that plays an important role in inhibiting T-cell proliferation and IL-2 secretion as an independent immunoregulatory mechanism (Dong et al, 1996, 1997). Selective mechanical studies in immune cells identify caveolin-1 as a functional receptor for mDPP-4 (Ohnuma et al, 2004; Hiromura et al, 2018). This is notable due to diverse activation pathways associated with CD26-caveolin-1-mediated signaling in different types of immune cells. On one hand, in T cells, ligation of mDPP-4 by caveolin-1 recruits CARMA1 and induces T-cell proliferation and NF-κB activation in a TCR-CD3-dependent manner (Ohnuma et al, 2007). On the other hand, in APCs, binding to mDPP-4 leads to disengagement from caveolin-1 of Toll-interacting protein (Tollip) and interleukin-1 receptor-associated serine/threonine kinase 1 (IRAK-1) with subsequent IRAK-1-induced upregulation of CD86 expression, thereby mobilizing the immune response (Ohnuma et al, 2004). A blockade of the mDPP-4–caveolin-1 signaling axis by fusion protein caveolin-1-Ig leads to anergy in CD4+ T-cells (Ohnuma et al, 2009).

1. Restoring paracrine crosstalk in the islet through facilitating GABAergic transmission in the islets

Menegaz and colleagues were able to establish that the islets of patients with T1D exhibit a significant reduction in GABA levels, which is not caused by a deficiency in GAD65 expression in β-cells, suggesting that loss of GABAergic signaling mechanisms may strongly correlate with T1D pathogenesis (Menegaz et al, 2019). They have, for the first time, elegantly identified the exact mechanisms of GABA release from the cytosol via volume regulatory anion channels (VRAC) in a pulsatile fashion independent of glucose concentration (Fig. 1) (Menegaz et al, 2019). As mentioned above, GABAergic transmission is a key β-cell mechanism for glucagon regulation and suppression. As GABA is continuously secreted by the β-cell, upon GSIS, the secreted insulin initiates downstream signaling through IR in the α-cells, leading to PI3K-AKT regulated assembly and translocation of GABAA receptors to α-cell membrane. GABAA receptors are chloride channels; once activated by extracellular available GABA, they allow chloride influx and hyperpolarize α-cell membrane shutting voltage-gated calcium channels, thereby inhibiting glucagon release (Fig. 3) (Xu et al, 2006). Other groups have also demonstrated protective and regenerative properties of GABA on the β-cells (Tian et al, 2013, 2017; Purwana et al, 2014). A recent study using a combination of orally administered GABA with alum-formulated human recombinant GAD65 (GAD65 Alum) in newly diagnosed T1D pediatric patients (GABA: 41 patients, GABA+GAD65 Alum: 25 patients, placebo: 31 patients) showed a significant reduction in fasting and meal-stimulated glucagon levels compared with control (Martin et al, 2022). The authors noted a high safety profile and good tolerability of GABA administration in exclusively pediatric patients and speculated on suitability for higher dosages for increased drug efficacy in future studies.

2. GABAergic signaling in immune cells

Dionisio and colleagues describe a comprehensive GABAergic system in lymphocytes that regulates activation and stimulus responses in T-cells (Dionisio et al, 2011). GABA uptake by the cells is directly dependent on receptor activation, which may be caused by ambient GABA gradients in physiological compartments or by secreted GABA by an autocrine or paracrine loop. Earlier studies also show that 100nM and higher concentrations of GABA inhibit T-cell proliferation and have an immunomodulatory effect by means of interaction with GABAA channels (Bjurstöm et al, 2008). More recent research on antitumor immunity that can be considered for autoimmune conditions also shows that activated B-cells secrete GABA, which directly promotes monocyte differentiation into IL-10-producing anti-inflammatory macrophages that inhibit CD8+ T-cell killer function (Zhang et al, 2021). Bhandage and colleagues have conducted an extensive investigation on the role of GABA in regulating immune cells in relation to T1D. They found that GABA successfully inhibited 16 of 26 cytokines that were significantly elevated in the plasma of T1D patients, as observed in cell assays (Bhandage et al, 2018). Importantly, the authors note that GABAA receptors in the brain seldom contain the ρ2 subunit, which is most prevalent in human peripheral blood mononuclear cells (PBMCs) presenting a specific therapeutic target for autoimmune diseases such as T1D. This research on T cells from T1D patients also reveals that depending on concentration, GABA regulates the release of up to 47 cytokines from human PBMCs and CD4+ T cells (Fig. 4). This aligns with previous research that has shown the inhibitory effects of GABA on several cytokines commonly associated with T1D manifestation and progression, such as IL-2 (Tian et al, 2004), interferon gamma (Tian et al, 2004), IL-6, IL-12 (Reyes-García et al, 2019), IL-1β (Zhao et al, 2014), and TNF-α (Swaroop et al, 2012).

Under these conditions, identifying novel applications of extrasynaptic GABAA and GABAB receptor modulators in autoimmune disease might be beneficial for investigating the impact of GABAergic signaling on inflammation and the control of immune cells. Treatment with lesogaberan, a peripherally restricted GABAB receptor agonist alone and in combination with anti-CD3 increased remission and rapidly inhibited β-cell destruction in NOD mice (Tian et al, 2021). In anti-CD3-stimulated human PBMCs, GABAAR was found to be responsible for inhibiting Ca2+ influx and NF-kB transcriptional activity (Alam et al, 2006; Prud’homme et al, 2013). As an example, diazepam, a positive allosteric modulator of GABAA receptors, successfully inhibited interferon gamma production in anti-CD3 stimulated human and murine CD4+ and CD8+ T cells (Wei et al, 2010; Soltani et al, 2011) and was shown to inhibit innate and adaptive immune responses in a number of animal studies (Sanders et al, 2015; Falcón et al, 2021). Evidently, GABA signaling plays an important role in regulating immune cell behaviors and cytokine release. As research results on GABA efficacy in T1D remain inconsistent, it is possible that other molecular pathways need to be activated for GABA to be sufficiently produced by pancreatic cells and subsequently reabsorbed by the immune cells in conditions of inflammation, cytokine assault, and glucotoxicity, associated with T1D.