Jul 2026· Journal of Immunology· Vol 215· 0 citations
TL;DR
Preliminary experiments revealed that both the nTregs and eTregs can suppress the cytotoxic function and cytokine secretion of the islet-specific CTLs, indicating that suppression is mediated by a cell contact-dependent mechanism.
Abstract
Type 1 diabetes (T1D) is an autoimmune disease characterized by the destruction of β-cells in the pancreatic islets, resulting in insulin deficiency. Currently the best treatment option for T1D is exogenous insulin, which regulates blood glucose levels, but does not change the underlying disease. The increasing number of T1D diagnoses highlight the need for novel therapies. An area of growing interest is the use of regulatory T cells (Tregs) to suppress autoreactive T cells. Clinical trials involving the transfer of autologous ex vivo expanded polyclonal CD4+ Tregs have shown this approach to be safe; however, it has limitations. These include challenges with ex vivo Treg expansion, antigen specificity, loss of immunosuppressive activity, and inherent Treg functional defects due to genetic deficiencies in autoimmune patients.
Our group has developed a patented protocol for generating engineered Tregs (eTregs) by expressing two transcription factors critical for Treg function, FOXP3 and Helios in conventional T cells. We hypothesize that our FOXP3+Helios+ eTregs can suppress the islet-specific reactivity of cytotoxic CD8+ T cells (CTLs). To test this hypothesis, we have generated CTLs specific for an islet-specific antigen and FOXP3+Helios+ eTregs, as well as isolated natural Tregs (nTregs), all from the same donor. We then compared the ability of the eTregs with that of nTregs to suppress CTL reactivity against the human β cell line βlox5.
Preliminary experiments revealed that both the nTregs and eTregs can suppress the cytotoxic function and cytokine secretion of the islet-specific CTLs. Separating the Tregs and CTLs using transwell inserts abrogated the suppressive effect, indicating that suppression is mediated by a cell contact-dependent mechanism.
These data indicate that FOXP3+Helios+ engineered Tregs can effectively suppress islet specific CTL responses in vitro and support the continued investigation of eTregs as a potential cell-based immunotherapy for T1D.
Breakthrough T1D (1-INO-2025-1709-A-N), NIH/NIGMS (P20GM130423), Children’s Mercy Hospital
Therapeutic Approaches to Autoimmunity (THER)
Type 1 diabetes (T1D) is a T cell-mediated autoimmune disease where CD8 T cells eliminate insulin-producing β cells in the pancreatic islets. Yet, MHC class II haplotypes confer the greatest genetic risk for the development of T1D, suggesting a critical role for CD4 T cells. Employing the non-obese diabetic (NOD) mouse model, our lab identified in the pancreatic lymph node (pLN) a stem-like β cell-specific CD8 T cell pool required to initiate and sustain disease: pLN β cell-specific stem-CD8 T cells self-renew and continuously give rise to differentiated progenies which migrate to the pancreas (PA) and eliminate β cells; the pLN stem-CD8 T cell pool is absolutely required to sustain β cell destruction. Given the importance of autoimmune stem-CD8 T cells and the association of MHC class II in T1D pathogenesis, we wanted to understand the role of CD4 T cells in autoimmune CD8 T cell stemness and differentiation.
We employed the NOD model and longitudinally assessed the phenotypic and functional characteristics of β cell-specific CD4 T cells using flow cytometry, serial transplantation, CRISPR/Cas9-mediated gene editing, and transcriptomic studies.
Our studies reveal, for the first time, how CD4 T cells drive autoimmune CD8 T cell stemness, differentiation, and pathogenicity. We find β cell-specific CD4 T cells in pLN and PA reveal two distinct populations based on their expression of TCF1, a transcription factor critical for stemness and self-renewal. Functional studies identify pLN TCF1hi CD4 T cells as stem-T cell subset needed to drive the generation and maintenance of autoimmune stem-CD8 T cells in pLN, their differentiation into β cell-destroying cytotoxic effector cells, and ultimately T1D.
A unique population of β cell-specific CD4 T cells in pLN is critical for autoimmune CD8 T cell stemness, differentiation and disease. Identifying therapeutic strategies that target autoimmune stem-CD4 T cells could emerge as powerful approaches for the treatment of T1D.
NIH grant 1F31DK145180, NIH grant R01AI173249, Juvenile Diabetes Research Foundation grant JDRF SRA-2023-1410-S-B,
Basic Autoimmunity (BA)
Ian McBain, P. Zumbo, S. Miakicheva et al.· Journal of Immunology· 0 citations
Type 1 diabetes (T1D) is an autoimmune disorder caused by T cell-mediated destruction of pancreatic β cells. There is a critical need to develop novel therapies for this disease, which requires further understanding of T1D pathogenesis. One pathway that is implicated is signaling through the immune receptor NKG2D. However, NKG2D’s role has remained unclear due to conflicting reports in the literature.
We generated multiple novel mouse strains on the non-obese diabetic (NOD) background to better define the role of NKG2D in autoimmune diabetes. We compared diabetes incidence, insulitis, including digital spatial profiling, and islet-specific CD8+ CTL generation between mice genetically deficient in NKG2D (NKG2D KO) and wild-type (WT) mice housed in both specific-pathogen-free (SPF) and germ-free (GF) conditions.
NKG2D KO mice had delayed diabetes, insulitis, and CD8+ T cell pancreatic infiltration, with the most significant effect in GF and male mice. Further, we found that NKG2D signaling did not occur in the pancreas, that NKG2D ligands were expressed in the pancreatic lymph node, that deletion of NKG2D in CD8+ T cells was required to delay diabetes, that there was reduced differentiation of CTL from islet-specific naive NKG2D KO CD8+ T cells, and that the transcriptome of NKG2D KO CTL was altered. Additionally, we found that blocking NKG2D signaling similarly reduced human CTL differentiation.
The results of our studies definitively demonstrate that NKG2D in CD8+ T cells plays a critical role in autoimmune diabetes. We show this role is to enhance CTL generation rather than the CTL effector response in islets as previously proposed. Further, our data explain the conflicting reports surrounding NKG2D’s role, as we reveal the existence of redundant, compensatory signaling that is influenced by the microbiome and sex. This inherent redundancy strongly suggests that signaling through NKG2D or its compensatory pathway is critical for islet-specific CTL generation.
Breakthrough T1D (3-SRA-2023-1412-S-B and 1-INO-2022-1118-A-N), NIH P20GM104936
Basic Autoimmunity (BA)
M. Markiewicz, Zoe K Bedrosian, Allison Manning et al.· Journal of Immunology· 0 citations
Type 1 diabetes (T1D) continues to be a complex, multifactorial autoimmune condition characterized by targeted destruction of pancreatic β-cells and permanent insulin dependence. How tremendous the change has been since the advent of insulin therapy, yet T1D patients remain at catastrophic risk from acute complications and chronic vascular injury, calling for more potent disease-modifying therapies. Developments in immunology and genetics have unraveled the interaction of environmental trigger events, including viral infection and gut microbiota, with genetic susceptibility, i.e., HLA class II alleles, on the aberration of immune tolerance and induction of β- cell autoimmunity. The last few decades have seen remarkable advances in the pathogenesis of T1D, including the discovery of β-cell autoantibodies and the pivotal role of autoreactive T cells. However, therapeutic trials in humans of immunomodulatory interventions, such as cyclosporine, anti-CD3 antibodies, and tolerogenic dendritic cells, have, to date, shown only temporary preservation of native insulin secretion, with safety limitations or failure to sustain efficacy being significant limitation. In parallel, efforts at inducing antigen-specific tolerance and microbiota modification have been promising in preclinical models but are still to be validated in humans. Technological advances, including continuous glucose monitoring and artificial pancreas systems, have enhanced glycemic management and quality of life without influencing causative autoimmunity. New strategies, β-cell replacement by transplant or xenograft, genetic engineering to favor immune evasion, and gene therapy for insulin production promise more definitive cures, but much remains in the path of safety, immune rejection, and long-term efficacy. Finally, in the years to come, the management of T1D will be a blend of early diagnosis, customized immunomodulation, improved sensing of glucose, and regeneration. Research on the genetic, immunologic, and environmental determinants for T1D and the creation of safer and more potent therapies will continue to be needed to advance beyond disease symptomatology management to disease modification and prevention.
Jasvinder Saini, Dushyant, Jagdeep Singh et al.· Current Topics in Medicinal...· 0 citations
Tregs play a key role in establishing and maintaining immune tolerance and homeostasis but despite progress, current Treg therapies face challenges of unstable phenotypes, lack of IL-2 support, and tissue specificity.
We present a strategy to address these challenges by harnessing a gene editing platform with dual AAV to engineer human regulatory T cells (EngTreg) from bulk CD4+ T cells resulting in the stable expression of FOXP3 and a chemically inducible cytokine signaling complex providing IL-2 signaling support. Additionally, a novel hypoimmune approach improves allogeneic cell persistence by preventing T, B, and NK cell mediated rejection, making possible an off-the-shelf approach. This modular engineering allows for indication specific, interchangeable tissue specific targeting including TCRs, CARs, or inflammation tuned alarmin receptors.
We present transcriptomic and flow cytometry analyses showing EngTregs express higher levels of core Treg and FOXP3 synergy genes, stability markers and tolerogenic proteins compared to cultured Tregs. EngTregs have been tailored for multiple indications: GNTI-122 targets IGRP for T1D, showing strong bystander suppression and disease prevention in mouse models. CAR19 EngTregs target B-cell driven autoimmune diseases with better safety compared to CAR-T cells. Gut epithelium targeting EngTregs ameliorate disease in murine IBD models. Tissue EngTregs target alarmin mediated inflammation and show efficacy in models of lung injury, AKI, and stroke.
Together, these data support EngTregs as a potent, stable, and versatile cell therapy platform for autoimmune, inflammatory, and ischemic diseases.
NIH-SBIR, venture capital, foundation funding
Translational and Interventional Immunology (TI)
P. Zarin, Gene I. Uenishi, Martina Sassone-Corsi et al.· Journal of Immunology· 0 citations
Engineered mesenchymal stem cells overexpressing AAT and gene delivery strategies provide an effective way to achieve sustained and localized AAT expression, and are expected to overcome the current challenges in clinical translation.
Underperformance and dysfunction of CD4+ regulatory T cells (Tregs) has been implicated in the pathogenesis of many autoimmune diseases. As such, multiple approaches toward Treg immunotherapy are being developed, most of which require ex vivo expansion and/or manipulation of Tregs, and in vivo strategies remain challenging to accomplish. Here, we hypothesized that constitutively high expression of the high-affinity IL2 receptor on Tregs could be exploited to target selective uptake of mRNA-LNPs by Tregs, enabling the potential development of an in vivo immunotherapy platform.
IL2 or αCD25 was conjugated to LNPs containing N1-Methylpseudouridine-substituted mRNA for eGFP using a SATA-maleimide based strategy. In vivo experiments were done using 6-week-old female C57BL/6 mice given 5 µg of LNP intravenously. In vitro experiments were performed using normal donor human splenocytes or PBMCs. Targeting was assessed using eGFP expression and flow cytometry.
To define the optimal method to target Tregs with mRNA-LNPs, we first compared αCD25 vs IL-2 conjugated LNPs. IL2-LNPs demonstrated superior targeting in vivo of splenic Tregs compared to αCD25-LNPs in both frequency (69.7% vs. 41.9% eGFP+ respectively, p = 0.03) and expression (1399 vs. 424 eGFP MFI, p = 0.01) 24 hours after delivery. IL2-LNP targeted Tregs expressing eGFP were present in spleen, lymph node, and blood 24 hours post-treatment (40-70% eGFP+) and remained detectable for at least 7 days. Little background uptake of IL2-LNPs was observed in other immune cell subsets. Finally, IL2-LNPs also delivered mRNA effectively to FoxP3+Tregs in vitro in human splenocytes in a dose-dependent manner with little uptake by other cell subsets.
Altogether, these data demonstrate that IL2-LNPs are an efficient and effective method of targeting Tregs in situ. Future studies will apply this powerful tool to transiently enhance and alter Treg function in vivo for interventional and therapeutic strategies in autoimmune disease models.
Breakthrough T1D (3-SRA-2024-1612-S-B)
Therapeutic Approaches to Autoimmunity (THER)
Erin Maule, Amie Albertus, Vladimir Shuvaev et al.· Journal of Immunology· 1 citation
