Reduced function of the adaptor SH2B3 promotes T1D via altered gc cytokine-regulated, T cell intrinsic immune tolerance.

Genome-wide association studies have identified SH2B3 as an important non-MHC gene for islet autoimmunity and type 1 diabetes (T1D). In this study, we found a single SH2B3 haplotype significantly associated with increased risk for human T1D, and this haplotype carries the single nucleotide variant rs3184504*T in SH2B3. To better characterize the role of SH2B3 in T1D, we used mouse modeling and found a T cell-intrinsic role for SH2B3 regulating peripheral tolerance. SH2B3 deficiency had minimal effect on TCR signaling or proliferation across antigen doses, yet enhanced cell survival and cytokine signaling including common gamma chain-dependent and interferon-gamma receptor signaling. SH2B3 deficient CD8+T cells showed augmented STAT5-MYC and effector-related gene expression partially reversed with blocking autocrine IL-2 in culture. Using the RIP-mOVA model, we found CD8+ T cells lacking SH2B3 promoted early islet destruction and diabetes without requiring CD4+ T cell help. SH2B3-deficient cells demonstrated increased survival post-transfer compared to control cells despite a similar proliferation profile in the same host. Next, we created a spontaneous NOD .Sh2b3 -/- mouse model and found markedly increased incidence and accelerated T1D across sexes. Collectively, these studies identify SH2B3 as a critical mediator of peripheral T cell tolerance limiting the T cell response to self-antigens. Article Highlights: The rs3184504 polymorphism, encoding a hypomorphic variant of the negative regulator SH2B3, strongly associates with T1D.SH2B3 deficiency results in hypersensitivity to cytokines, including IL-2, in murine CD4+ and CD8+ T cells.SH2B3 deficient CD8+ T cells exhibit a comparable transcriptome to wild-type CD8+ T cells at baseline, but upon antigen stimulation SH2B3 deficient cells upregulate genes characteristic of enhanced JAK/STAT signaling and effector functions.We found a T-cell intrinsic role of SH2B3 leading to severe islet destruction in an adoptive transfer murine T1D model, while global SH2B3 deficiency accelerated spontaneous NOD diabetes across sexes.

Activated PI3Kδ Specifically Perturbs Mouse Regulatory T Cell Homeostasis and Function Leading to Immune Dysregulation.

FOXP3+ regulatory T cells (Treg) are required for maintaining immune tolerance and preventing systemic autoimmunity. PI3Kδ is required for normal Treg development and function. However, the impacts of dysregulated PI3Kδ signaling on Treg function remain incompletely understood. In this study, we used a conditional mouse model of activated PI3Kδ syndrome to investigate the role of altered PI3Kδ signaling specifically within the Treg compartment. Activated mice expressing a PIK3CD gain-of-function mutation (aPIK3CD) specifically within the Treg compartment exhibited weight loss and evidence for chronic inflammation, as demonstrated by increased memory/effector CD4+ and CD8+ T cells with enhanced IFN-γ secretion, spontaneous germinal center responses, and production of broad-spectrum autoantibodies. Intriguingly, aPIK3CD facilitated Treg precursor development within the thymus and an increase in peripheral Treg numbers. Peripheral Treg, however, exhibited an altered phenotype, including increased PD-1 expression and reduced competitive fitness. Consistent with these findings, Treg-specific aPIK3CD mice mounted an elevated humoral response following immunization with a T cell-dependent Ag, which correlated with a decrease in follicular Treg. Taken together, these findings demonstrate that an optimal threshold of PI3Kδ activity is critical for Treg homeostasis and function, suggesting that PI3Kδ signaling in Treg might be therapeutically targeted to either augment or inhibit immune responses.

GNTI-122: an autologous antigen-specific engineered Treg cell therapy for type 1 diabetes.

Tregs have the potential to establish long-term immune tolerance in patients recently diagnosed with type 1 diabetes (T1D) by preserving ß cell function. Adoptive transfer of autologous thymic Tregs, although safe, exhibited limited efficacy in previous T1D clinical trials, likely reflecting a lack of tissue specificity, limited IL-2 signaling support, and in vivo plasticity of Tregs. Here, we report a cell engineering strategy using bulk CD4+ T cells to generate a Treg cell therapy (GNTI-122) that stably expresses FOXP3, targets the pancreas and draining lymph nodes, and incorporates a chemically inducible signaling complex (CISC). GNTI-122 cells maintained an expression profile consistent with Treg phenotype and function. Activation of CISC using rapamycin mediated concentration-dependent STAT5 phosphorylation and, in concert with T cell receptor engagement, promoted cell proliferation. In response to the cognate antigen, GNTI-122 exhibited direct and bystander suppression of polyclonal, islet-specific effector T cells from patients with T1D. In an adoptive transfer mouse model of T1D, a mouse engineered-Treg analog of GNTI-122 trafficked to the pancreas, decreased the severity of insulitis, and prevented progression to diabetes. Taken together, these findings demonstrate in vitro and in vivo activity and support further development of GNTI-122 as a potential treatment for T1D.

A chemically inducible IL-2 receptor signaling complex allows for effective in vitro and in vivo selection of engineered CD4+ T cells.

Engineered T cells represent an emerging therapeutic modality. However, complex engineering strategies can present a challenge for enriching and expanding therapeutic cells at clinical scale. In addition, lack of in vivo cytokine support can lead to poor engraftment of transferred T cells, including regulatory T cells (Treg). Here, we establish a cell-intrinsic selection system that leverages the dependency of primary T cells on IL-2 signaling. FRB-IL2RB and FKBP-IL2RG fusion proteins were identified permitting selective expansion of primary CD4+ T cells in rapamycin supplemented medium. This chemically inducible signaling complex (CISC) was subsequently incorporated into HDR donor templates designed to drive expression of the Treg master regulator FOXP3. Following editing of CD4+ T cells, CISC+ engineered Treg (CISC EngTreg) were selectively expanded using rapamycin and maintained Treg activity. Following transfer into immunodeficient mice treated with rapamycin, CISC EngTreg exhibited sustained engraftment in the absence of IL-2. Furthermore, in vivo CISC engagement increased the therapeutic activity of CISC EngTreg. Finally, an editing strategy targeting the TRAC locus permitted generation and selective enrichment of CISC+ functional CD19-CAR-T cells. Together, CISC provides a robust platform to achieve both in vitro enrichment and in vivo engraftment and activation, features likely beneficial across multiple gene-edited T cell applications.

Activated PI3Kδ specifically perturbs mouse Treg homeostasis and function leading to immune dysregulation.

Foxp3+ regulatory T cells (Treg) are required for maintaining immune tolerance and preventing systemic autoimmunity. PI3Kδ is required for normal Treg development and function. However, the impacts of dysregulated PI3Kδ signaling on Treg function remain incompletely understood. Here, we used a conditional mouse model of activated PI3Kδ syndrome (APDS) to investigate the role of altered PI3Kδ signaling specifically within the Treg compartment. Aged mice expressing a PIK3CD gain-of-function mutation (aPIK3CD) specifically within the Treg compartment exhibited weight loss and evidence for chronic inflammation as demonstrated by increased memory/effector CD4+ and CD8+ T cells with enhanced IFN-γ secretion, spontaneous germinal center responses and production of broad-spectrum autoantibodies. Intriguingly, aPIK3CD facilitated Treg precursor development within the thymus and an increase in peripheral Treg numbers. Peripheral Treg, however, exhibited an altered phenotype including increased PD1 expression and reduced competitive fitness. Consistent with these findings, Treg specific-aPIK3CD mice mounted an elevated humoral response following immunization with a T-cell dependent antigen, that correlated with a decrease in follicular Treg. Taken together, these findings demonstrate that an optimal threshold of PI3Kδ activity is critical for Treg homeostasis and function, suggesting that PI3Kδ signaling in Treg might be therapeutically targeted to either augment or inhibit immune responses.

Pancreatic islet-specific engineered Tregs exhibit robust antigen-specific and bystander immune suppression in type 1 diabetes models.

Adoptive transfer of regulatory T cells (Tregs) is therapeutic in type 1 diabetes (T1D) mouse models. Tregs that are specific for pancreatic islets are more potent than polyclonal Tregs in preventing disease. However, the frequency of antigen-specific natural Tregs is extremely low, and ex vivo expansion may destabilize Tregs, leading to an effector phenotype. Here, we generated durable, antigen-specific engineered Tregs (EngTregs) from primary human CD4+ T cells by combining FOXP3 homology-directed repair editing and lentiviral T cell receptor (TCR) delivery. Using TCRs derived from clonally expanded CD4+ T cells isolated from patients with T1D, we generated islet-specific EngTregs that suppressed effector T cell (Teff) proliferation and cytokine production. EngTregs suppressed Teffs recognizing the same islet antigen in addition to bystander Teffs recognizing other islet antigens through production of soluble mediators and both direct and indirect mechanisms. Adoptively transferred murine islet-specific EngTregs homed to the pancreas and blocked diabetes triggered by islet-specific Teffs or diabetogenic polyclonal Teffs in recipient mice. These data demonstrate the potential of antigen-specific EngTregs as a targeted therapy for preventing T1D.

Islet allografts expressing a PD-L1 and IDO fusion protein evade immune rejection and reverse preexisting diabetes in immunocompetent mice without systemic immunosuppression.

Allogeneic islet transplantation is a promising experimental therapy for poorly controlled diabetes. Despite pharmacological immunosuppression, long-term islet engraftment remains elusive. Here, we designed a synthetic fusion transgene coupling PD-L1 and indoleamine dioxygenase [hereafter PIDO] whose constitutive expression prevents immune destruction of genetically engineered islet allograft transplanted in immunocompetent mice. PIDO expressing murine islets maintain robust dynamic insulin secretion in vitro and when transplanted in allogeneic hyperglycemic murine recipients reverse pre-existing streptozotocin-induced and autoimmune diabetes in the absence of pharmacological immunosuppression for more than 50 and 8 weeks, respectively, and is dependent on host CD4 competence. Additionally, PIDO expression in allografts preserves endocrine functional viability of islets and promotes a localized tolerogenic milieu characterized by the suppression of host CD8 T cell and phagocyte recruitment and accumulation of FOXP3+ Tregs. Furthermore, in the canine model of xenogeneic islet transplantation, muscle implanted PIDO-expressing porcine islets displayed physiological glucose-responsive insulin secretion competency in euglycemic recipient for up to 20 weeks. In conclusion, the PIDO transgenic technology enables host CD4+ T cell-modulated immune evasiveness and long-term functional viability of islet allo- and xenografts in immune-competent recipients without the need for pharmacological immune suppression and would allow for improved outcomes for tissue transplantation.

Pancreatic Stellate Cells Prolong Ex Vivo Islet Viability and Function and Improve Engraftment.

Preserving islet health and function is critical during pretransplant culture to improve islet transplantation outcome and for ex vivo modeling of diabetes for pharmaceutical drug discovery. The limited islet engraftment potential is primarily attributable to loss of extracellular matrix (ECM) support and interaction. Multipotent cells with ECM depositing competency improve islet survival during short coculture period. However, role of pancreatic stellate cells (PSCs) and their ECM support in preserving ex vivo islet physiology remains largely unknown. Here, we report novel cytoprotective effects of culture-adapted porcine PSCs and role of their ECM-mediated intercellular communication on pig, mouse and human islets ex vivo. Using direct-contact coculture system, we demonstrate that porcine PSCs preserve and significantly prolong islet viability and function from 7 ± 3 days to more than 28 ± 5 (P < .001) days in vitro. These beneficial effects of PSCs on islet health are not species-specific. Using NSC47924 to specifically inhibit 37/67 kDa laminin receptor (LR), we identified that LR-mediated intercellular communication is essential for PSCs to protect functional viability of islets in vitro. Finally, our results demonstrate that PSC co-transplantation improved function and enhanced capacity of syngeneic islets to reverse hyperglycemia in mice with preexisiting diabetes. Cumulatively, our findings unveil novel effects of culture-adapted PSCs on islet health likely mirroring in vivo niche interaction. Furthermore, islet and PSC coculture may aid in development of ex vivo diabetes modeling and also suggests that a combined islet-PSC tissue engineered implant may significantly improve islet transplantation outcome.